Vaccine peptide combinations against cat allergy

ABSTRACT

The present invention relates to compositions comprising peptides for preventing or treating allergy to cats, and in particular to optimal combinations of peptides

FIELD OF THE INVENTION

The present invention relates to compositions comprising peptides forpreventing or treating allergy to cats, and in particular to optimalcombinations of peptides

BACKGROUND OF THE INVENTION

T-cell antigen recognition requires antigen presenting cells (APCs) topresent antigen fragments (peptides) on their cell surface inassociation with molecules of the major histocompatibility complex(MHC). T cells use their antigen specific T-cell receptors (TCRs) torecognise the antigen fragments presented by the APC. Such recognitionacts as a trigger to the immune system to generate a range of responsesto eradicate the antigen which has been recognised.

Recognition of external antigens by the immune system of an organism,such as man, can in some cases result in diseases, known as atopicconditions. Examples of the latter are the allergic diseases includingasthma, atopic dermatitis and allergic rhinitis. In this group ofdiseases, B lymphocytes generate antibodies of the IgE class (in humans)which bind externally derived antigens, which are referred to in thiscontext as allergens since these molecules elicit an allergic response.Production of allergen-specific IgE is dependent upon T lymphocyteswhich are also activated by (are specific for) the allergen.Allergen-specific IgE antibodies bind to the surface of cells such asbasophils and mast cells by virtue of the expression by these cells ofsurface receptors for IgE.

Crosslinking of surface bound IgE molecules by allergen results indegranulation of these effector cells causing release of inflammatorymediators such as histamine, 5-hydroxtryptamine and lipid mediators suchas the sulphidoleukotrienes. In addition to IgE-dependent events,certain allergic diseases such as asthma are characterised byIgE-independent events.

Allergic IgE-mediated diseases are currently treated with agents whichprovide symptomatic relief or prevention. Examples of such agents areanti-histamines, β2 agonists, and glucocorticosteroids. In addition,some IgE-mediated diseases are treated by desensitisation proceduresthat involve the periodic injection of allergen components or extracts.Desensitisation treatments may induce an IgG response that competes withIgE for allergen, or they may induce specific suppressor T cells thatblock the synthesis of IgE directed against allergen. This form oftreatment is not always effective and poses the risk of provokingserious side effects, particularly general anaphylactic shock. This canbe fatal unless recognised immediately and treated with adrenaline. Atherapeutic treatment that would decrease or eliminate the unwantedallergic-immune response to a particular allergen, without altering theimmune reactivity to other foreign antigens or triggering an allergicresponse itself would be of great benefit to allergic individuals.

Approximately 10% of the worlds human population are allergic to cats(Felis domesticus) and up to 67% of asthmatic patients are sensitive tocat allergens. The major allergen produced by cats is the glycoproteinFel d1, which elicits a response in 90-95% of patients suffering fromcat allergy. A therapeutic or preventative treatment would therefore beof great benefit to humans that suffer or are at risk of suffering fromcat allergy.

SUMMARY OF THE INVENTION

The present inventors have discovered that certain combinations ofpeptide fragments of the Fel d1 protein are particularly useful indesensitising individuals to Fel d1 allergen. The polypeptidecombinations of the invention have been selected for their ability tobind to many MHC Class II molecules, and cause T cell proliferation withminimal histamine release. The compositions, products, vectors andformulations of the invention may therefore be provided to individualsfor preventing or treating allergy to cats by tolerisation.

The polypeptides of the invention were initially selected as potential Tcell epitopes through use of peptide-MHC binding assays. See for exampleFIG. 1 which demonstrates the ability of a range of peptides derivedfrom Fel d1 chains 1 and 2 to bind to multiple DR types in MHC class IIbinding assays These candidate polypeptides were then further screenedfor potential use in tolerisation.

A difficulty associated with approaches to desensitisation based onpeptide immunisation lies in how to select an appropriate size andregion of the allergen as the basis for the peptide to be used forimmunisation. The size of the peptide of choice is crucial. If thepeptide is too small, the vaccine would not be effective in inducing animmunological response. If the peptides are too large, or if the wholeantigen is introduced into an individual, there is the risk of inducingadverse reactions, such as anaphylaxis, which may be fatal.

The polypeptides of the invention have been selected to retain T cellspecificity whilst being small enough in size to not possess significanttertiary structure that would enable them to retain the conformation ofan IgE-binding epitope of the whole molecule. The polypeptides of theinvention therefore do not induce significant crosslinking of adjacentspecific IgE molecules on cells such as mast cells and basophils andconsequently do not cause significant histamine release.

The peptides of the invention are advantageous in that uponadministration to a sample of T cells they result in T cellproliferation whilst causing minimal histamine release. This isdemonstrated in Example 2. The polypeptides of the inventions arecapable of inducing a late phase response in a cat allergic individual.The composition, products and formulations of the invention comprisingthese polypeptides or polynucleotides that are capable of expressingthese polypeptides are therefore useful and effective in reducinghypersensitivity to Fel d1 allergen in individuals that are sensitisedto this allergen.

A further advantage of the invention is the ability of the combinationsof peptides to broadly target Major Histocompatibility Complex (MHC)molecules. T cell receptors (TCRs) are highly variable in theirspecificity. Variability is generated, as with antibody molecules,through gene recombination events within the cell. TCRs recogniseantigen in the form of short peptides bound to molecules encoded by thegenes of the Major Histocompatibility Complex (MHC). These gene productsare the same molecules that give rise to “tissue types” used intransplantation and are also referred tows Human Leukocyte Antigenmolecules (HLAs) which terms may be used interchangeably. Individual MHCmolecules possess peptide binding grooves which, due to their shape andcharge are only capable of binding a limited group of peptides. Thepeptides bound by one MHC molecule may not necessarily be bound by otherMHC molecules.

When a protein molecule such as an antigen or allergen is taken up byantigen presenting cells such as B lymphocytes, dendritic cells,monocytes and macrophages, the molecule is enzymatically degraded withinthe cell. The process of degradation gives rise to peptide fragments ofthe molecule which, if they are of the appropriate size, charge andshape, may then bind within the peptide binding groove of certain MHCmolecules and be subsequently displayed upon the surface of antigenpresenting cells. If the peptide/MHC complexes are present upon theantigen presenting cell surface in sufficient numbers they may thenactivate T cells which bear the appropriate peptide/MHC-specific T cellreceptors.

Due to the polymorphic nature of the MHC, individuals in an outbredpopulation such as man will express different combinations of MHCmolecules on their cell surfaces. Since different MHC molecules can binddifferent peptides from the same molecule based on the size, charge andshape of the peptide, different individuals will display a differentrepertoire of peptides bound to their MHC molecules. Identification ofuniversal MHC-binding peptide epitopes in an outbred population such asman is more difficult than in inbred animals (such as certain strains oflaboratory mice). On the basis of differential MHC expression betweenindividuals and the inherent differences in peptide binding andpresentation which this brings, it is unlikely that a single peptide canbe identified which will be of use for desensitisation therapy in man.

The peptide combination of the invention, however, provides a broadcoverage of efficacy over the human population by targeting the majorityof the population's MHC. It would not, for example, be necessary to typethe patient or individual to determine which MHC Class II molecules heor she possesses in order to determine what peptide or combination ofpeptides would be effective. A vaccine formulated with the peptides ofthe invention would therefore have broad utility.

The inventors' work has produced peptide combinations with the followingcharacteristics:

-   -   the combination binds to many different MHC Class II molecules        (see FIG. 2 which shows the large number of combinations that do        not bind to many different MHC molecules)    -   the combinations produce the same or less histamine release than        the whole allergen and/or have a cytokine release profile        equivalent to the whole allergen    -   the peptides of the combinations are soluble.

Accordingly, the present invention provides a composition for use inpreventing or treating allergy to cats by tolerisation comprising:

-   -   a) four or more polypeptides selected from any of SEQ ID NO: 1,        2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and optionally    -   b) one, two or three polypeptides having the following        characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 not            selected in a); and        -   (ii) 9 to 30 amino acids in length.

Preferably, the composition of the invention comprises either:

-   -   (i) at least two peptides which exhibit strong binding and at        least one peptide which exhibits moderate binding to each member        of a panel of HLA molecules; or    -   (ii) at least one peptide which exhibits strong binding and at        least two peptides which exhibit moderate binding to each member        of said panel of HLA molecules;        wherein the panel of HLA molecules comprises at least seven        different HLA molecules encoded by different alleles which have        a cumulative frequency in an outbred human population of at        least 80%; and/or    -   (iii) wherein the composition is capable of inducing histamine        release in a sample from a cat allergic individual at a level        which is no higher than 5% above the histamine release induced        in a sample from the same individual by whole Fel d 1 allergen;        and/or    -   (iv) wherein the composition induces a cytokine release profile        in a PBMC sample from a cat allergic individual which is        equivalent to the cytokine release profile in a sample from the        same individual induced by whole Fel d 1 allergen.

Typically the outbred human population is Caucasian, and/or the panel ofHLA molecules comprises at least HLA-DR1, DR3, DR4, DR7, DR11, DR13 andDR15; and optionally also comprises HLA-DRB4 and DRB5.

DESCRIPTION OF THE DRAWINGS

FIG. 1—Peptides derived from Fel d1 chains 1 and 2 were tested forability to bind to multiple DR types in MHC class II binding assays.Peptides that showed promiscuous binding characteristics were selectedand combined to generate mixtures of peptides that bind to a broadpopulation of MHC class II types.

FIGS. 2A-2Q—Graphical representations of peptide mixtures showing thosewhich bind to a broad population of MHC class II types.

FIG. 3—Proliferation: percentage responders and quality of response.FIG. 3 summarises proliferative responses to peptides and antigens. Thepercentage of individuals mounting a detectable proliferative responseis shown in the black bars. Grey (weak), white (moderate) and hashed(strong) bars provide a breakdown of the quality of these responses.Quality is arbitrarily defined by Stimulation Index (SI: ratio of countsin the presence of antigen/peptide divided by counts in medium alone).Thus for peptide 1 (MLA01), 12% of subjects made a proliferativeresponse and of these 92% were weak, none were moderate and 8% werehigh. Proliferative responses to individual peptides/antigens werevariable (black bar). 92% of subjects had positive proliferativeresponses to the positive control antigen PPD. The majority of thesewere strong responses (hashed bar). 75% of subjects responded to catdander extract, with 59% of the responses (i.e. 59% of the 75%) beingweak. The response to the mixture of 7 preferred peptides (SEQ ID NOS: 1TO 7) was almost identical to cat dander extract (CAT).

FIG. 4—Percentage of responders by cytokine. FIG. 4 summarises thepercentage of individuals who mounted a detectable response to each ofthe peptides/antigens by production of the three cytokines measured. Thepositive control antigen PPD elicited a cytokine production in almostall individuals (IFN-γ: 91%, IL-13: 97% and IL-10: 96%). Whole catallergen and the mixture of 7 peptides elicited a cytokine response inapproximately 80% or more of subjects. Individual peptides elicitedresponses of differing frequency. In general cytokine productionappeared to be a more sensitive method of detecting responses withlarger percentages of individuals giving positive cytokine responsesthan proliferative responses. In most cases, IL-10 secretion wasdetected in the largest number of subjects and IFN-γ detected leastfrequently.

FIG. 5—Percentage of individuals producing IFN-γ and strength ofresponse following cell culture with peptide/antigen. IFN-γ responseswere detected in 26-44% of subjects in response to individual peptides.These responses were predominantly very low to low to moderate. Complexantigens induced more frequent responses (peptide mixture 80%, catdander 79%, PPD 91%). These responses were low to moderate to high. PPDresponses were particularly high (89 of PPD responses were above 100pg/ml).

FIG. 6—Percentage of individuals producing IL-13 and strength ofresponse following cell culture with peptide/antigen. IL-13 responseswere detected in between 33-68% of subjects in response to individualpeptides. These responses were predominantly very low to low, although asignificant number of moderate responses were detected. This may reflectthe Th2 nature of allergic sensitisation in these subjects. Complexantigens induced more frequent responses (peptide mixture 85%, catdander 93%, PPD 97%). These responses were low to moderate to high.

FIG. 7—Percentage of individuals producing IL-10 and strength ofresponse following cell culture with peptide/antigen. IL-10 responseswere detected in between 46-75% of subjects in response to individualpeptides. These responses were predominantly very low to low. Complexantigens induced more frequent responses (peptide mixture 93%, catdander 96%, PPD 96%). These responses were low to moderate. Very few“high” IL-10 responses were observed.

FIG. 8—A representative plot showing the average LPSR area before andafter treatment for all eight patients in the 12.0 nmol cohort of theclinical trial of a preferred mixture of peptides of the invention.

DESCRIPTION OF THE SEQUENCES MENTIONED HEREIN

SEQ ID NO: 1 to 16 provide the polypeptide sequences of the invention.SEQ ID NOS: 1 to 16 correspond to peptides MLA01, MLA03, MLA04, MLA05,MLA07, MLA12, MLA14, MLA02, MLA06, MLA11, MLA15, MLA16, MLA08, MLA09,MLA10 and MLA13 respectively as shown in the Examples and FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a composition for use in preventing or treatingallergy to cats by tolerisation comprising:

-   -   a) four or more polypeptides selected from any of SEQ ID NO: 1,        2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and optionally    -   b) one, two or three polypeptides having the following        characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 contiguous amino acids in any of SEQ            ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 not selected            in a); and        -   (ii) 9 to 30 amino acids in length.

The invention also provides products and formulations comprising thepolypeptides of the invention and compositions, products and vectorscomprising polynucleotides capable of expressing the polypeptides of theinvention for use in preventing or treating cat allergy by tolerisation.

Peptide Fragments of Fel d1 Protein

The major allergen produced by the domestic cat Felis catus (Felisdomesticus) is the glycoprotein Fel d1. This 39 kDa protein is formedfrom two 17 kDa subunits, each consisting of two disulphide-linkedpeptides (Fel d1 Chain 1 and Chain 2). The amino acid sequence of Fel d1is disclosed in WO 91/06571. The major source of the Fel d1 protein isthe sebaceous glands, although expression is also detected in salivaryglands and the anal glands. The function of the Fel d1 protein iscurrently unknown, although it is possibly a pheromone binding protein.

The peptides of the invention are derived from Fel d1. The terms“peptide” and “polypeptide” are used interchangeably herein. Fel d1 isalso referred to herein as “the allergen”.

The composition of the invention comprises four or more polypeptidesselected from any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.Optionally, the composition may comprise one, two or three furtherpolypeptides. These further polypeptides relate to (i.e. are typicallyhomologues and/or fragments of) the other sequences, i.e. SEQ ID NO 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, that are not amongst the four ormore polypeptides already selected. The one, two or three furtherpolypeptides may be identical to any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12. The composition may therefore comprise four, five,six or seven different polypeptides as provided in any of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. However, the optional one, two orthree further polypeptides do not need to be 100% identical to any ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. They are preferablyat least 65% identical to at least 9 or more contiguous amino acids inany of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, not alreadyselected for the four or more polypeptides.

In other words, the invention provides a composition for use in theprevention or treatment of cat allergy by tolerisation comprising a)four or more polypeptides selected from any one of the following aminoacid sequences:

(SEQ ID NO: 1) CPAVKRDVDLFLT; (SEQ ID NO: 2) EQVAQYKALPVVLENA; (SEQ IDNO: 3) KALPVVLENARILKNCV; (SEQ ID NO: 4) RILKNCVDAKMTEEDKE; (SEQ ID NO:5) KENALSLLDKIYTSPL; (SEQ ID NO: 6) TAMKKIQDCYVENGLI; (SEQ ID NO: 7)SRVLDGLVMTTISSSK; (SEQ ID NO: 8) LFLTGTPDEYVEQVAQY; (SEQ ID NO: 9)KMTEEDKENALSLLDK; (SEQ ID NO: 10) LTKVNATEPERTAMKK; (SEQ ID NO: 11)ISSSKDCMGEAVQNTV; (SEQ ID NO: 12) AVQNTVEDLKLNTLGRAnd optionally, the composition may comprise b) one, two or threefurther polypeptides having the following characteristics:

-   -   (i) comprising sequence having at least 65% sequence identity to        at least 9 or more contiguous amino acids in any of SEQ ID NO: 1        to 12 above not selected in a); and    -   (ii) 9 to 30 amino acids in length.

The invention also provides a product containing a) four or morepolypeptides selected from any one of the following amino acidsequences:

(SEQ ID NO: 1) CPAVKRDVDLFLT; (SEQ ID NO: 2) EQVAQYKALPVVLENA; (SEQ IDNO: 3) KALPVVLENARILKNCV; (SEQ ID NO: 4) RILKNCVDAKMTEEDKE; (SEQ ID NO:5) KENALSLLDKIYTSPL; (SEQ ID NO: 6) TAMKKIQDCYVENGLI; (SEQ ID NO: 7)SRVLDGLVMTTISSSK; (SEQ ID NO: 8) LFLTGTPDEYVEQVAQY; (SEQ ID NO: 9)KMTEEDKENALSLLDK; (SEQ ID NO: 10) LTKVNATEPERTAMKK; (SEQ ID NO: 11)ISSSKDCMGEAVQNTV; (SEQ ID NO: 12) AVQNTVEDLKLNTLGRand optionally, the product may comprise b) one or more furtherpolypeptides having the following characteristics:

-   -   (i) comprising sequence having at least 65% sequence identity to        at least 9 or more contiguous amino acids in any of SEQ ID NO: 1        to 12 above not selected in a); and    -   (ii) 9 to 30 amino acids in length,        wherein each different polypeptide is for simultaneous, separate        or sequential use in the prevention or treatment of cat allergy        by tolerisation.

In more detail therefore, the invention provides a product containing:

-   -   (a) A polypeptide selected from any one of SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 or 12;    -   (b) A polypeptide selected from any one of SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 or 12, that is not selected in (a)        above;    -   (c) A polypeptide selected from any one of SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 or 12, that is not selected in (a)        or (b) above;    -   (d) A polypeptide selected from any one of SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 or 12, that is not selected in (a), (b)        or (c) above; and optionally    -   (e) A polypeptide having the following characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1 to 12 not selected in a), b), c) or d)            above; and        -   (ii) 9 to 30 amino acids in length; and optionally    -   (f) A polypeptide fragment of Fel d1 protein having the        following characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1 to 12 not selected in a), b), c), d) or e)            above; and        -   (ii) 9 to 30 amino acids in length; and optionally    -   (g) A polypeptide fragment of Fel d1 protein having the        following characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1 to 12 not selected in a), b), c), d), e)            or f) above; and        -   (ii) 9 to 30 amino acids in length;            for simultaneous, separate or sequential use in the            prevention or treatment of cat allergy by tolerisation.

The composition or products of the invention may therefore comprisevariants of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.Peptide fragments according to the invention may be derived bytruncation, e.g. by removal of one or more amino acids from the N and/orC-terminal ends of a polypeptide. Fragments may also be generated by oneor more internal deletions, provided that the core 9 amino acids thatmakes up the T cell epitope is not substantially disrupted.

For example, a variant of SEQ ID NO: 1 may comprise a fragment of SEQ IDNO: 1, i.e. a shorter sequence. This may include a deletion of one, two,three or four amino acids from the N-terminal end of SEQ ID NO: 1 orfrom the C-terminal end of SEQ ID NO: 1. Such deletions may be made fromboth ends of SEQ ID NO: 1. A variant of SEQ ID NO: 1 may includeadditional amino acids (for example from the cat Fel d1 proteinsequence) extending beyond the end(s) of SEQ ID NO: 1. A variant mayinclude a combination of the deletions and additions discussed above.For example, amino acids may be deleted from one end of SEQ ID NO: 1,but additional amino acids from the full length Fel d1 protein sequencemay be added at the other end of SEQ ID NO: 1. The same discussion ofvariants above also applies to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11and 12.

A variant peptide may include one or more amino acid substitutions fromthe amino acid sequence of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12, or a fragment thereof. A variant peptide may comprisesequence having at least 65% sequence identity to at least 9 or morecontiguous amino acids in any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12. More preferably a suitable variant may comprise at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% amino acid identity to at least 9 contiguous aminoacids of any of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Thislevel of amino acid identity may be seen at any section of the peptide,although it is preferably the core region. The level of amino acididentity is over at least 9 contiguous amino acids but it may be atleast 10, 11, 12, 13, 14, 15 or at least 16 or 17 amino acids, dependingon the size of the peptides of comparison. Accordingly, any of theabove-specified levels of identity may be across the entire length ofsequence.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10; Multiple alignmentparameters—Matrix: PAM, Gap open penalty: 10.00, % identity for delay:30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix:no, Gap extension penalty: 0.20, Residue-specific gap penalties: on,Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequenceidentity at a particular residue is intended to include identicalresidues which have simply been derivatized.

A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 aminoacid substitutions from any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12. Substitution variants preferably involve the replacement ofone or more amino acids with the same number of amino acids and makingconservative amino acid substitutions. For example, an amino acid may besubstituted with an alternative amino acid having similar properties,for example, another basic amino acid, another acidic amino acid,another neutral amino acid, another charged amino acid, anotherhydrophilic amino acid, another hydrophobic amino acid, another polaramino acid, another aromatic amino acid or another aliphatic amino acid.Some properties of the 20 main amino acids which can be used to selectsuitable substituents are as follows:

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, Asp polar, hydrophilic,neutral charged (−) Pro hydrophobic, neutral Glu polar, hydrophilic, Glnpolar, hydrophilic, charged (−) neutral Phe aromatic, hydrophobic, Argpolar, hydrophilic, neutral charged (+) Gly aliphatic, neutral Serpolar, hydrophilic, neutral His aromatic, polar, hydrophilic, Thr polar,hydrophilic, neutral charged (+) Val aliphatic, hydrophobic, Ilealiphatic, hydrophobic, neutral neutral Lys polar, hydrophilic,charged(+) Trp aromatic, hydrophobic, Leu aliphatic, hydrophobic,neutral neutral Tyr aromatic, polar, hydrophobic

Further variants include those in which instead of the naturallyoccurring amino acid the amino acid which appears in the sequence is astructural analog thereof. Amino acids used in the sequences may also bemodified, e.g. labelled, providing the function of the peptide is notsignificantly adversely affected. Where the peptide has a sequence thatvaries from the sequence of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 or a fragment thereof, the substitutions may occur acrossthe full length of the sequence, within the sequence of any of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or outside the sequence ofany of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. For example,the variations described herein, such as additions, deletions,substitutions and modifications, may occur within the sequence of any ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. A variant peptidemay comprise or consist essentially of the amino acid sequence of any ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 in which one, two,three, four or more amino acid substitutions have been made. A variantpeptide may comprise a fragment of Fel d1 that is larger than any of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In this embodiment, thevariations described herein, such as substitutions and modifications,may occur within and/or outside the sequence of any of SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

The variant peptides of the invention are 9 to 30 amino acids in lengthinclusive. Preferably, they may be from 9 to 20 or more preferably 13 to17 amino acids in length. The peptides may be the same length as thepeptide sequences in any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12.

The peptides may be chemically derived from the polypeptide allergen,for example by proteolytic cleavage or can be derived in an intellectualsense from the polypeptide allergen, for example by making use of theamino acid sequence of the polypeptide allergen and synthesisingpeptides based on the sequence. Peptides may be synthesised usingmethods well known in the art.

The term “peptide” includes not only molecules in which amino acidresidues are joined by peptide (—CO—NH—) linkages but also molecules inwhich the peptide bond is reversed. Such retro-inverso peptidomimeticsmay be made using methods known in the art, for example such as thosedescribed in Meziere et al (1997) J. Immunol. 159, 3230-3237. Thisapproach involves making pseudopeptides containing changes involving thebackbone, and not the orientation of side chains. Meziere et al (1997)show that, at least for MHC class II and T helper cell responses, thesepseudopeptides are useful. Retro-inverse peptides, which contain NH—CObonds instead of CO—NH peptide bonds, are much more resistant toproteolysis.

Similarly, the peptide bond may be dispensed with altogether providedthat an appropriate linker moiety which retains the spacing between thecarbon atoms of the amino acid residues is used; it is particularlypreferred if the linker moiety has substantially the same chargedistribution and substantially the same planarity as a peptide bond. Itwill also be appreciated that the peptide may conveniently be blocked atits N-or C-terminus so as to help reduce susceptibility toexoproteolytic digestion. For example, the N-terminal amino group of thepeptides may be protected by reacting with a carboxylic acid and theC-terminal carboxyl group of the peptide may be protected by reactingwith an amine. Other examples of modifications include glycosylation andphosphorylation. Another potential modification is that hydrogens on theside chain amines of R or K may be replaced with methylene groups(—NH₂→—NH(Me) or —N(Me)₂).

Analogues of peptides according to the invention may also includepeptide variants that increase or decrease the peptide's half-life invivo. Examples of analogues capable of increasing the half-life ofpeptides used according to the invention include peptoid analogues ofthe peptides, D-amino acid derivatives of the peptides, andpeptide-peptoid hybrids. A further embodiment of the variantpolypeptides used according to the invention comprises D-amino acidforms of the polypeptide. The preparation of polypeptides using D-aminoacids rather than L-amino acids greatly decreases any unwanted breakdownof such an agent by normal metabolic processes, decreasing the amountsof agent which needs to be administered, along with the frequency of itsadministration.

The peptides provided by the present invention may be derived fromsplice variants of Fel d1 encoded by mRNA generated by alternativesplicing of the primary transcripts encoding the Fel d1 chains. Thepeptides may also be derived from amino acid mutants, glycosylationvariants and other covalent derivatives of Fel d1 which retain at leastan MHC-binding property of the allergen. Exemplary derivatives includemolecules wherein the peptides of the invention are covalently modifiedby substitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid. Further included arenaturally occurring variants of Fel d1 found in different cats. Such avariant may be encoded by an allelic variant or represent an alternativesplicing variant.

Variants as described above may be prepared during synthesis of thepeptide or by post-production modification, or when the peptide is inrecombinant form using the known techniques of site-directedmutagenesis, random mutagenesis, or enzymatic cleavage and/or ligationof nucleic acids.

In accordance with the invention, the further one, two or three peptidesthat the composition may comprise are preferably functional variants ofany of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. That is, thepeptides are preferably capable of inducing an immune response. Inparticular, they are capable of inducing a late phase response in a catallergic individual. This may be tested by the ability of the peptide toinduce T cell proliferation in a sample of T cells. Methods of testingthe induction of T cell proliferation are well known in the art and onesuch method is exemplified in Example 2. Preferably the one or morefurther peptides are capable of causing T cell proliferation in at least20% of samples of T cells, wherein each sample is obtained fromdifferent cat allergic individuals in the population. The compositionsof the invention are preferably capable of inducing T cell proliferationin 30% or more samples of T cells obtained from of a panel of catallergic individuals. More preferably, the compositions are capable ofinducing T cell proliferation in 35% or more, 40% or more, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% or more of samples obtainedfrom sensitized individuals in a panel. The number of individuals in apanel of cat allergic individuals may be any number greater than one,for example at least 2, 3, 5, 10, 15, 20, 30, 50, 80, or at least 100individuals. It is preferred if the peptides cause T cell proliferation,but do not lead to the release of histamine from enriched basophils ormast cell preparations from a sensitised individual. There may be somehistamine release, but preferably the composition does not causesignificantly more histamine release than a composition comprising the 7different polypeptides shown in SEQ ID NO: 1 to 7.

Suitable variants capable of binding to TCRs may be derived empiricallyor selected according to known criteria. Within a single peptide thereare certain residues which contribute to binding within the MHC antigenbinding groove and other residues which interact with hypervariableregions of the T cell receptor (Allen et al (1987) Nature 327: 713-5).

Within the residues contributing to T cell receptor interaction, ahierarchy has been demonstrated which pertains to dependency of T cellactivation upon substitution of a given peptide residue. Using peptideswhich have had one or more T cell receptor contact residues substitutedwith a different amino acid, several groups have demonstrated profoundeffects upon the process of T cell activation. Evavold & Allen (1991)Nature 252: 1308-10) demonstrated the dissociation of T cellproliferation and cytokine production. In this in vitro model, a T cellclone specific for residues 64-76 of haemoglobin (in the context ofI-E^(k)), was challenged with a peptide analogue in which a conservativesubstitution of aspartic acid for glutamic acid had been made. Thissubstitution did not significantly interfere with the capacity of theanalogue to bind to I-E^(k).

Following in vitro challenge of a T cell clone with this analogue, noproliferation was detected although IL-4 secretion was maintained, aswas the capacity of the clone to help B cell responses. In a subsequentstudy the same group demonstrated the separation of T cell-mediatedcytolysis from cytokine production. In this instance, the formerremained unaltered while the latter was impaired. The efficacy ofaltered peptide ligands in vivo was initially demonstrated in a murinemodel of EAE (experimental allergic encephalomyelitis) by McDevitt andcolleagues (Smilek et al (1991) Proc Natl Acad Sci USA 88: 9633-9637).In this model EAE is induced by immunisation with the encephalitogenicpeptide Ac1-11 of MBP (myelin basic protein). Substitution at positionfour (lysine) with an alanine residue generated a peptide which boundwell to its restricting element (Aα^(u)Aβ^(u)), but which wasnon-immunogenic in the susceptible PL/JxSJLF1 strain and which,furthermore prevented the onset of EAE when administered either beforeor after immunisation with the encephalitogenic peptide. Thus, residuescan be identified in peptides which affect the ability of the peptidesto induce various functions of T-cells.

Advantageously, peptides may be designed to favour T-cell proliferationand induction of desensitisation. Metzler and Wraith have demonstratedimproved tolerogenic capacity of peptides in which substitutionsincreasing peptide-MHC affinity have been made (Metzler & Wraith (1993)Int Immunol˜: 1159-65). That an altered peptide ligand can causelong-term and profound anergy in cloned T cells was demonstrated bySloan-Lancaster et al (1993) Nature 363: 156-9.

The compositions of the invention are capable of inducing a late phaseresponse in an individual that is sensitised to Fel d1 allergen. Theterm “late phase response” includes the meaning as set forth in Allergyand Allergic Diseases (1997) A. B. Kay (Ed.), Blackwell Science, pp1113-1130. The late phase response may be any late phase response (LPR).Preferably, the peptides are capable of inducing a late asthmaticresponse (LAR) or a late rhinitic response, or a late phase skinresponse or a late phase ocular response. Whether or not a particularpeptide can give rise to a LPR can be determined using methods wellknown in the art; a particularly preferred method is that described inCromwell O, Durham S R, Shaw R J, Mackay J and Kay A B. Provocationtests and measurements of mediators from mast cells and basophils inasthma and allergic rhinitis. In: Handbook of Experimental Immunology(4) Chapter 127, Editor: Weir D M, Blackwell Scientific Publications,1986.

Thus, preferably, the individual peptides of the invention are able toinduce a LPR in an individual who has been sensitised to Fel d1allergen. Whether or not an individual has been sensitised to theallergen may be determined by well known procedures such as skin pricktesting with solutions of allergen extracts, induction of cutaneousLPRs, clinical history, allergen challenge and radioallergosorbent test(RAST) for measurement of allergen specific IgE. Whether or not aparticular individual is expected to benefit from treatment may bedetermined by the physician based, for example, on such tests.

Desensitising or tolerising an individual to Fel d1 allergen meansinhibition or dampening of allergic tissue reactions induced by Fel d1in appropriately sensitised individuals. It has been shown that T cellscan be selectively activated, and then rendered unresponsive. Moreoverthe anergising or elimination of these T-cells leads to desensitisationof the patient for a particular allergen. The desensitisation manifestsitself as a reduction in response to an allergen or allergen-derivedpeptide, or preferably an elimination of such a response, on second andfurther administrations of the allergen or allergen-derived peptide. Thesecond administration may be made after a suitable period of time haselapsed to allow desensitisation to occur; this is preferably any periodbetween one day and several weeks. An interval of around two weeks ispreferred.

Although the compositions of the invention are able to induce a LPR in acat allergic individual, it should be appreciated that when acomposition is used to treat a patient it is preferable that asufficiently low concentration of the composition is used such that noobservable LPR will occur but the response will be sufficient topartially desensitise the T cells such that the next (preferably higher)dose may be given, and so on. In this way the dose is built up to givefull desensitisation but often without ever inducing a LPR in thepatient. Although, the composition or peptide is able to do so at ahigher concentration than is administered.

The compositions of the invention preferably are capable of inducing alate phase response in 50% or more of a panel of cat allergicindividuals from the population. More preferably, the compositions arecapable of inducing a LPR in 55% or more, 60% or more, 65% or more, 70%or more, 75% or more, 80% or more, 85% or more, or 90% or more ofsensitized individuals in a panel. Whether or not the compositions areable to induce a LPR in a certain percentage of a panel of subjects canbe determined by methods which are well known in the art.

Properties of Peptide Combinations MHC Binding

Preferred combinations of peptides typically bind to a large number ofdifferent HLA molecules. This is advantageous in that a largerproportion of individuals in a population will be tolerised by thecombination. Thus preferred combinations comprise either:

-   -   (iii) at least two peptides which exhibit strong binding and at        least one peptide which exhibits moderate binding to each member        of a panel of HLA molecules; or    -   (iv) at least one peptide which exhibits strong binding and at        least two peptides which exhibit moderate binding to each member        of said panel of HLA molecules;        wherein the panel of HLA molecules comprises at least seven        different HLA molecules encoded by different alleles which have        a cumulative frequency in an outbred human population of at        least 80%, or at least 85%, 90%, 95% or 99%.

Strength of MHC binding may be evaluated by any suitable method.Preferred methods include competitive inhibition assays wherein bindingis measured relative to a reference peptide. The reference peptide istypically a peptide which is known to be a strong binder for a given MHCmolecule. In such an assay, a peptide is a weak binder for a given HLAmolecule if it has an IC50 more than 100 fold lower than the referencepeptide for the given HLA molecule. A peptide is a moderate binder is ithas an IC50 more than 20 fold lower but less than a 100 fold lower thanthe reference peptide for the given HLA molecule. A peptide is a strongbinder if it has an IC50 less than 20 fold lower than the referencepeptide for the given HLA molecule.

The outbred human population may be any population, typically aCaucasian population. The panel of HLA molecules typically comprises atleast HLA-DR1, DR3, DR4, DR7, DR11, DR13 and DR15; and optionally alsocomprises HLA-DRB4 and DRB5. Suitable reference peptides for these HLAmolecules are:

DR1 (DRB1*0101 allele): HA 306-318 (PKYVKQNTLKLAT); DR3 (DRB1*0301allele): MT216 (AKTIAYDEEARRGLE); DR4 (DRB1*0401 allele): HA 306-318(PKYVKQNTLKLAT); DR7 (DRB1*0701 allele): YKL (AAYAAAKAAALAA); DR 11(DRB1*1101 allele): HA 306-318 (PKYVKQNTLKLAT); DR13 (DRB1*1301 allele):B1 21-36 (TERVRLVTRHIYNREE); DR15 (DRB1*1501 allele): A3 152-166(EAEQLRRAYLDGTGVE); DRB4 (DRB4*0101 allele): E2/E7 (AGDLLAIETDKATT); andDRB5 (DRB5*0101 allele): HA 306-318 (PKYVKQNTLKLAT).

Histamine Release

Preferred combinations of peptides typically induce histamine release ina sample from a cat allergic individual containing basophils or mastcells, which is no higher than 5%, 6%, 7%, 8%, 9% or 10% greater thanthe histamine release induced in a sample from the same individual orpopulation of individuals by the whole Fel d 1 allergen.

Most preferably, the combination induces histamine release which is nohigher than 5%, 6%, 7%, 8%, 9% or 10% greater than the histamine releaseinduced in a sample from the same individual or population ofindividuals by a composition comprising the 7 different polypeptidesshown in SEQ ID NO: 1 to 7.

A sample from a cat allergic individual is typically a sample ofperipheral blood mononuclear cells (PBMCs) which may be prepared as isstandard in the art. An example of a suitable method involves isolationof PBMCs from a heparinised blood sample obtained from a subject. PBMC'sare typically isolated from such a sample by density gradientseparation.

Histamine release may be assessed by any suitable method, for example byELISA. A number of suitable assay kits are commercially available totest levels of histamine release from cells in response to any givenhistamine release agent. Typically, a sample of approximately 5×10⁵ to5×10⁶ PBMCs will be incubated with a given histamine release agent at agiven concentration. Histamine concentration in the incubation medium ora sample of the incubation medium will measured at the end of theincubation. Incubation is typically for 30 minutes at 37° C.

Where the histamine release agent is a peptide or combination ofpeptides it will typically be administered at a number of differentdilutions within a concentration range comparable to that which would beexpected to be present in vivo. For example, a 10 mg dose of a singlepeptide entering a blood volume of 5 litres would result in a bloodconcentration of 2 ng/ml (2×10⁻⁶ mg/ml). Thus, a suitable concentrationrange for a peptide or combination of peptides is typically 10 mg/ml to1 ng/ml. Single, duplicate or triplicate measurement may be made at eachtested dilution within said range. Approximately 5×10⁵ PBMCs aretypically required for each measurement. Suitable positive controls willalso be tested at appropriate concentrations which may be readilydetermined by the skilled person. Suitable positive controls includewhole Fel d 1allergen or a suitable alternative such as commerciallyavailable whole cat dander extract. Spontaneous histamine release by asample of cells which is not treated with a histamine release agent mayalso be measured as a negative control/indicator of background histaminerelease. Where two or more dilutions of a peptide/allergen preparationelicit 10% or more histamine release above background, or where a singlevalue of 10% or more above background is achieved at the highestconcentration tested, this will typically be considered a “positivehistamine release”.

The histamine concentration in the incubation medium of any sample willtypically be measured by ELISA. Suitable ELISA assays typically involveadding a histamine acylation agent to a sample of the incubation mediumtogether with a suitable buffer. Acylated histamine is more stable thanhistamine and samples treated in this way may be stored for longer priorto analysis. Analysis typically involves the addition ofalkaline-phosphatase conjugated anti-acyl-histamine reagents, followedby the addition of a suitable chromogenic alkaline-phosphatasesubstrate. Histamine concentration is determined by measurement ofabsorbance and comparison to a standard curve calibrated against knownhistamine concentrations.

Cytokine Release

Preferred combinations of peptides typically induce a cytokine releaseprofile in a sample from a cat allergic individual containing T cells,which is equivalent to the cytokine release profile induced in a samplefrom the same individual or population of individuals by the whole Fel d1 allergen.

Most preferably, the combination induces a cytokine release profile in asample from a cat allergic individual or population of individualscontaining T cells, which is equivalent to the cytokine release profileinduced in a sample from the same individual or population by acomposition comprising the 7 different polypeptides shown in SEQ ID NO:1 to 7.

A sample from a cat allergic individual or population is typically asample of peripheral blood mononuclear cells (PBMCs) which may beprepared as is standard in the art. Cytokine release profile may beassessed by any suitable method. Suitable methods include measuring thelevel of one, two, three or more different cytokines released in asample in independent assays. Suitable assays include ELISA and Luminexassays.

A cytokine release profile induced in one sample is considered to beequivalent to the cytokine release profile of a different sample whenthe level of certain specific cytokines produced is similar in bothsamples. More specifically, the cytokine release profiles of twodifferent samples are considered to be equivalent when the levels ofIL-10 and IL-13 produced in one sample differ by no more 5%, 6%, 7%, 8%,9% or 10% from the levels of IL-10 and IL-13 produced in the secondsample.

Thus, a preferred peptide combination induces production of IL-10 andIL-13 at levels which differ by no more than 10% from the levels ofIL-10 and IL-13 induced in a sample from the same individual orpopulation of individuals by the whole Fel d 1 allergen.

A typical cytokine release assay is as follows:

250 μl of a 200 μg/ml solution of the appropriate antigen or peptideconcentration is distributed into the appropriate wells of, for example,48 well plates. Plates are then incubated in a humidified 5% CO₂incubator at 37° C. for a maximum of 4 hours. 250 μl of a 5×10⁶ cell/mlPBMC suspension is then added to each well and the plates returned tothe incubator for 5 days. Samples of culture supernatant are thenharvested as multiple aliquots for use in ELISA assays. The samples maybe frozen and stored prior to analysis. One aliquot is tested for thepresence of one cytokine. Typically the presence of a cytokine isestablished using an ELISA assay according to practices standard in theart. The cytokine concentrations in a sample are typically determined byinterpolation from standard curves generated in the same assay.

Nucleic Acids and Vectors

The individual peptides that make up the compositions and products ofthe invention may be administered directly, or may be administeredindirectly by expression from an encoding sequence. For example, apolynucleotide may be provided that encodes a peptide of the invention,such as any of the peptides described above. A peptide of the inventionmay thus be produced from or delivered in the form of a polynucleotidewhich encodes, and is capable of expressing, it. Any reference herein tothe use, delivery or administration of a peptide of the invention isintended to include the indirect use, delivery or administration of sucha peptide via expression from a polynucleotide that encodes it.

Accordingly, the invention provides a composition for use in preventingor treating allergy to cats by tolerisation comprising four or moredifferent polynucleotide sequences which when expressed cause theproduction of a composition for use in preventing or treating allergy tocats by tolerisation comprising:

a) four or more polypeptides selected from any of SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12; and optionally

b) one, two or three polypeptides having the following characteristics:

-   -   (i) comprising sequence having at least 65% sequence identity to        at least 9 or more contiguous amino acids in any of SEQ ID NO:        1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 not selected in a); and    -   (ii) 9 to 30 amino acids in length.

The invention also provides a product for use in preventing or treatingallergy to cats by tolerisation containing:

-   -   a) four or more polynucleotides capable of expressing a        different polypeptide selected from SEQ ID NO: 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11 or 12, and optionally    -   b) one, two or three polynucleotides capable of expressing        different polypeptides having the following characteristics:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 not            selected in a); and        -   (ii) 9 to 30 amino acids in length,            wherein each different polypeptide is for simultaneous,            separate of sequential use in the prevention or treatment of            allergy to cats in a human.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or purified form. A nucleic acidsequence which “encodes” a selected polypeptide is a nucleic acidmolecule which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Sambrook et al(1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

The polynucleotide molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the peptide of the invention in vivo in a targeted subject. Theseexpression cassettes, in turn, are typically provided within vectors(e.g., plasmids or recombinant viral vectors) which are suitable for useas reagents for nucleic acid immunization. Such an expression cassettemay be administered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a peptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Thus, the present invention provides avector for use in preventing or treating allergy to cats by tolerisationcomprising four or more polynucleotide sequences which encode differentpolypeptides of the invention and optionally one or more furtherpolynucleotide sequences which encode different polypeptides as definedherein. The vector may comprise 4, 5, 6, 7, 8, 9, 10, 11 or 12polynucleotide sequences which encode different polypeptides of theinvention.

Furthermore, it will be appreciated that the compositions and productsof the invention may comprise a mixture of polypeptides andpolynucleotides. Accordingly, the invention provides a composition orproduct as defined herein, wherein in place of any one of thepolypeptide is a polynucleotide capable of expressing said polypeptide.

Expression vectors are routinely constructed in the art of molecularbiology and may for example involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow forexpression of a peptide of the invention. Other suitable vectors wouldbe apparent to persons skilled in the art. By way of further example inthis regard we refer to Sambrook et al.

Thus, a polypeptide of the invention may be provided by delivering sucha vector to a cell and allowing transcription from the vector to occur.Preferably, a polynucleotide of the invention or for use in theinvention in a vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by thehost cell, i.e. the vector is an expression vector.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given regulatory sequence, such as a promoter,operably linked to a nucleic acid sequence is capable of effecting theexpression of that sequence when the proper enzymes are present. Thepromoter need not be contiguous with the sequence, so long as itfunctions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the nucleic acid sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

A number of expression systems have been described in the art, each ofwhich typically consists of a vector containing a gene or nucleotidesequence of interest operably linked to expression control sequences.These control sequences include transcriptional promoter sequences andtranscriptional start and termination sequences. The vectors of theinvention may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter. A“plasmid” is a vector in the form of an extrachromosomal geneticelement. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a resistance gene for a fungal vector. Vectors may be used invitro, for example for the production of DNA or RNA or used to transfector transform a host cell, for example, a mammalian host cell. Thevectors may also be adapted to be used in vivo, for example to allow invivo expression of the polypeptide.

A “promoter” is a nucleotide sequence which initiates and regulatestranscription of a polypeptide-encoding polynucleotide. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris repressed by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters. It is intended that the term “promoter” or“control element” includes full-length promoter regions and functional(e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the presentinvention may additionally comprise a signal peptide sequence. Thesignal peptide sequence is generally inserted in operable linkage withthe promoter such that the signal peptide is expressed and facilitatessecretion of a polypeptide encoded by coding sequence also in operablelinkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 aminoacids for example 15 to 20 amino acids. Often the amino acids arepredominantly hydrophobic. In a typical situation, a signal peptidetargets a growing polypeptide chain bearing the signal peptide to theendoplasmic reticulum of the expressing cell. The signal peptide iscleaved off in the endoplasmic reticulum, allowing for secretion of thepolypeptide via the Golgi apparatus. Thus, a peptide of the inventionmay be provided to an individual by expression from cells within theindividual, and secretion from those cells.

Alternatively, polynucleotides of the invention may be expressed in asuitable manner to allow presentation of a peptide of the invention byan MHC class II molecule at the surface of an antigen presenting cell.For example, a polynucleotide, expression cassette or vector of theinvention may be targeted to antigen presenting cells, or the expressionof encoded peptide may be preferentially stimulated or induced in suchcells.

Polynucleotides of interest may be used in vitro, ex vivo or in vivo inthe production of a peptide of the invention. Such polynucleotides maybe administered or used in the prevention or treatment of allergy tocats by tolerisation.

Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule canbe introduced directly into the recipient subject, such as by standardintramuscular or intradermal injection; transdermal particle delivery;inhalation; topically, or by oral, intranasal or mucosal modes ofadministration. The molecule alternatively can be introduced ex vivointo cells that have been removed from a subject. For example, apolynucleotide, expression cassette or vector of the invention may beintroduced into APCs of an individual ex vivo. Cells containing thenucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the peptide encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

The polypeptides, polynucleotides, vectors or cells of the invention maybe present in a substantially isolated form. They may be mixed withcarriers or diluents which will not interfere with their intended useand still be regarded as substantially isolated. They may also be in asubstantially purified form, in which case they will generally compriseat least 90%, e.g. at least 95%, 98% or 99%, of the proteins,polynucleotides, cells or dry mass of the preparation.

Antigen Presenting Cells (APCs)

The invention encompasses the use in vitro of a method of producing apopulation of APCs that present the peptides of the invention on theirsurface, that may be subsequently used in therapy. Such a method may becarried out ex vivo on a sample of cells that have been obtained from apatient. The APCs produced in this way therefore form a pharmaceuticalagent that can be used in the treatment or prevention of cat allergy bytolerisation. The cells should be accepted by the immune system of theindividual because they derive from that individual. Delivery of cellsthat have been produced in this way to the individual from whom theywere originally obtained, thus forms a therapeutic embodiment of theinvention.

Formulations and Compositions

The peptides, polynucleotides, vectors and cells of the invention may beprovided to an individual either singly or in combination. Each moleculeor cell of the invention may be provided to an individual in anisolated, substantially isolated, purified or substantially purifiedform. For example, a peptide of the invention may be provided to anindividual substantially free from the other peptides.

Whilst it may be possible for the peptides, polynucleotides orcompositions according to the invention to be presented in raw form, itis preferable to present them as a pharmaceutical formulation. Thus,according to a further aspect of the invention, the present inventionprovides a pharmaceutical formulation for use in preventing or treatingallergy to cats by tolerisation comprising a composition, vector orproduct according to the invention together with one or morepharmaceutically acceptable carriers or diluents and optionally one ormore other therapeutic ingredients. The carrier (s) must be ‘acceptable’in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Typically,carriers for injection, and the final formulation, are sterile andpyrogen free. Formulation of a composition comprising the peptide,polynucleotides or cells of the invention can be carried out usingstandard pharmaceutical formulation chemistries and methodologies all ofwhich are readily available to the reasonably skilled artisan.

For example, compositions containing one or more molecules or cells ofthe invention can be combined with one or more pharmaceuticallyacceptable excipients or vehicles. Auxiliary substances, such as wettingor emulsifying agents, pH buffering substances and the like, may bepresent in the excipient or vehicle. These excipients, vehicles andauxiliary substances are generally pharmaceutical agents that do notinduce an immune response in the individual receiving the composition,and which may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, polyethyleneglycol, hyaluronic acid, glycerol andethanol. Pharmaceutically acceptable salts can also be included therein,for example, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients, vehicles andauxiliary substances is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectablecompositions may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi-dose containers containing apreservative. Compositions include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchcompositions may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a composition for parenteraladministration, the active ingredient is provided in dry (for e.g., apowder or granules) form for reconstitution with a suitable vehicle(e.g., sterile pyrogen-free water) prior to parenteral administration ofthe reconstituted composition. The pharmaceutical compositions may beprepared, packaged, or sold in the form of a sterile injectable aqueousor oily suspension or solution. This suspension or solution may beformulated according to the known art, and may comprise, in addition tothe active ingredient, additional ingredients such as the dispersingagents, wetting agents, or suspending agents described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono-or di-glycerides. Otherparentally-administrable compositions which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Alternatively, the peptides or polynucleotides of the present inventionmay be encapsulated, adsorbed to, or associated with, particulatecarriers. Suitable particulate carriers include those derived frompolymethyl methacrylate polymers, as well as PLG microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jefferyet al. (1993) Pharm. Res. 10:362-368. Other particulate systems andpolymers can also be used, for example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugatesof these molecules.

The formulation of any of the peptides, polynucleotides or cellsmentioned herein will depend upon factors such as the nature of thesubstance and the method of delivery. Any such substance may beadministered in a variety of dosage forms. It may be administered orally(e.g. as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules), parenterally, subcutaneously, byinhalation, intradermally, intravenously, intramuscularly,intrasternally, transdermally or by infusion techniques. The substancemay also be administered as suppositories. A physician will be able todetermine the required route of administration for each particularindividual.

The compositions of formulations of the invention will comprise asuitable concentration of each peptide/polynucleotide/cell to beeffective without causing adverse reaction. Typically, the concentrationof each peptide in the composition will be in the range of 0.03 to 200nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180nmol/ml, 5 to 75 nmol/ml or 10 to 50 nmol/ml. The composition orformulations should have a purity of greater than 95% or 98% or a purityof at least 99%.

Therapeutic Methods and Individual to be Treated

The present invention relates to peptides, polynucleotides, vectors andcells that are capable of desensitising or tolerising human individualsto Fel d1 allergen and are therefore useful in the prevention ortreatment of cat allergy. The invention provides compositions, products,vectors and formulations for use in preventing or treating allergy tocats by tolerisation. The invention also provides a method of tolerisingor desensitizing a cat allergic individual comprising administering,either singly or in combination the polypeptides/polynucleotides/cellsof the invention as described above.

The individual to be treated or provided with the composition orformulation of the invention is preferably human. It will be appreciatedthat the individual to be treated may be known to be sensitised to Feld1 allergy, at risk of being sensitised or suspected of beingsensitised. The individual can be tested for sensitisation usingtechniques well known in the art and as described herein. Alternatively,the individual may have a family history of allergy to cats. It may notbe necessary to test an individual for sensitisation to Fel d1 becausethe individual may display symptoms of allergy when brought intoproximity to a cat. By proximity is meant 10 metres or less, 5 metres orless, 2 metres or less, 1 metre or less, or 0 metres from the cat.Symptoms of allergy can include itchy eyes, runny nose, breathingdifficulties, red itchy skin or rash.

The individual to be treated may be of any age. However, preferably, theindividual may be in the age group of 1 to 90, 5 to 60, 10 to 40, ormore preferably 18 to 35. Groups of individuals that are likely tobenefit from the treatment are for example cat owners, veterinarians andother cat handlers.

Preferably, the individual to be treated is from a population that hasMHC allele frequencies within the range of frequencies that arerepresentative of the Caucasian population. Reference population allelefrequencies for 11 common DRB1 allele families are shown in Table 3 ofExample 2 (Data from HLA Facts Book, Parham and Barber). Referencefrequencies were obtained by analysis of multiple studies reportingfrequencies and the figures shown are mean values. Preferably therefore,the individual to be treated is from a population that has equivalentMHC allele frequencies as the reference population for the allelesreferred to Table 3 (such as for at least 1, 2, 3, 4, 5 or all of thealleles), for example within the ranges of those figures plus or minus1, 2, 3, 5, 10, 15 or 20%.

Preferably the individual is from a population where the allelefrequencies of the following DRB1 alleles is

4—at least 9%7—at least 10%11—at least 8%.

The individual may have had allergy to cat for at least 2 weeks, 1month, 6 months, 1 year or 5 years. The individual may suffer from arash, nasal congestion, nasal discharge and/or coughing caused by theallergy. The individual may or may not have been administered with othercompositions/compounds which treat cat allergy. The individual may livein a population comprising at least 0.1 cats per human habitant.

Diagnostic Method

The invention also provides a method of detecting whether an individualhas or is at risk of developing a disorder, wherein the disordercomprises allergic symptoms in response to cat allergen.

The individual is typically a mammal, preferably a human. The individualto be tested in the method is preferably between the ages of 1 year and80 years, more preferably between the ages of 1 year and 60, 50, 40, 30or 20 years, and most preferably between the ages of 1 year and 16years.

The individual may have been diagnosed or may be suspected of sufferingfrom a disorder which is classified as intrinsic or non-allergic, forexample, intrinsic or non-allergic asthma. The individual may lack adetectable antibody response to a cat allergen, in particular an IgEresponse to cat allergen. Suitable assays to detect IgE include thePharmacia™ CAP system. Using this system, the individual typicallyscores 0 or 0/1.

The individual may be a patient suffering from or diagnosed as sufferingfrom symptoms which are typically associated with allergy such as itchyeyes, runny nose, breathing difficulties, red itchy skin or rash, in theabsence of an identifiable trigger. The first occurrence or diagnosis ofthese symptoms may occur when the individual is older than 15 years ofage. For example, the individual may be at least 15, 16, 17, 18, 20, 22,24, 26, 28 or 30 years of age at the first occurrence or diagnosis ofsymptoms of allergy which are typically associated with allergy.

The method of the invention concerns determining whether an individualhas a T cell response to a cat allergen, in particular the major catallergen, Fel d 1. Such a T cell response will be present in catallergic individuals. Without being bound by any hypothesis, theinventors consider that intrinsic or non-allergic disorders are also infact caused by a T cell-driven, IgE independent immune response.Accordingly these disorders also have an allergen trigger, but it doesnot give rise to allergen-specific IgE. Rather, it gives rise to a Tcell response which can be characterised by T cell proliferation or therelease of cytokines. For example, the cytokines released may includeIL-5, which is involved in the recruitment of eosinophils. Accordingly,the T cell response can drive the induction of eosinophilic reactions inan individual.

Whether an individual has a T cell response to Fel d 1 is determined bymeasuring whether or not the individual has a T cell response to apeptide or combination of peptides according to the invention. Whetheror not the individual has such a response may be determined by anysuitable method, typically a method which can be used to detectproliferation of allergen-experienced T cells or the presence ofcytokine released by allergen-experienced T cells. A positive responseby the patient's T cells to the peptide or combination of the inventionindicates that the patient has or is more likely to develop allergy-likesymptoms in response to the allergen. A negative response indicates thatthe patient has allergy-like symptoms which are not caused by the catallergen, or is less likely to develop allergy-like symptoms in responseto the cat allergen.

The T cells which respond to the peptide or combination in the methodare generally T cells which have been pre-sensitised in vivo toallergen. These allergen-experienced T cells are generally present inthe peripheral blood of a individual, i.e. within the population ofperipheral blood mononuclear cells (PBMCs) in the individual. The Tcells may be CD4 and/or CD8 T cells.

In the method the T cells can be contacted with the peptide orcombination of the invention in vitro or in vivo, preferably in vitro ina sample from the individual.

Generally the T cells which are contacted in the method are taken fromthe individual in a blood sample, although other types of samples whichcontain T cells can be used. The sample may be added directly to theassay or may be processed first. Typically the processing may comprisestandard techniques such as gradient centrifugation to separate the Tcells, with resuspension in any suitable volume. Alternatively, theprocessing may comprise diluting of the sample, for example with water,buffer or media. The sample may be diluted from 1.5 to 100 fold, forexample 2 to 50 or 5 to 10 fold.

The processing may comprise separation of components of the sample.Typically mononuclear cells (MCs) are separated from the samples. TheMCs will comprise the T cells and antigen presenting cells (APCs). Thusin the method the APCs present in the separated MCs can present thepeptide to the T cells. In another embodiment only T cells, such as onlyCD4 T cells, can be purified from the sample. PBMCs, MCs and T cells canbe separated from the sample using techniques known in the art.

Preferably the T cells used in the assay are in the form of unprocessedor diluted samples, are freshly isolated T cells (such as in the form offreshly isolated MCs or PBMCs) which are used directly ex vivo, i.e.they are not cultured before being used in the method or are thawedcells (which were previously frozen). However the T cells can becultured before use, for example in the presence of the allergen, andgenerally also exogenous growth promoting cytokines. During culturingthe allergen is typically present on the surface of APCs, such as theAPC used in the method. Pre-culturing of the T cells may lead to anincrease in the sensitivity of the method. Thus the T cells can beconverted into cell lines, such as short term cell lines.

The APC which is typically present in the method may come from the sameindividual as the T cell or from a different individual. The APC may bea naturally occurring APC or an artificial APC. The APC is a cell whichis capable of presenting the antigen to a T cell. It is typically aB-cell, dendritic cell or macrophage. It is typically separated from thesame sample as the T cell and is typically co-purified with the T cell.Thus the APC may be present in MCs or PBMCs. The APC is typically afreshly isolated ex vivo cell or a cultured cell. It may be in the formof a cell line, such as a short term or immortalised cell line. The APCmay express empty MHC class II molecules on its surface.

In one embodiment the peptide or combination of the invention is addeddirectly to an assay comprising T cells and APCs. As discussed above theT cells and APCs in such an assay could be in the form of MCs.

In one embodiment the peptide or combination of peptides is provided tothe APC in the absence of the T cell. The APC is then provided to the Tcell, typically after being allowed to present the allergen on itssurface. The peptide or combination of peptides may have been taken upinside the APC and presented, or simply be taken up onto the surfacewithout entering inside the APC.

Typically 10⁵ to 10⁷, preferably 2.5×10⁵ to 10⁶ PBMCs are added to eachassay. In the case where the peptide or combination or peptides is addeddirectly to the assay it is typically added as a peptide with aconcentration from 10⁻¹ to 10³ μg/ml, preferably 0.5 to 50 μg/ml or 1 to10 μg/ml.

Typically the length of time for which the T cells are incubated withthe peptide or combination is from 4 to 24 hours (preferably 5 to 18hours) for effector T cells or for more than 24 hours for central memorycells. When using ex vivo PBMCs it has been found that 5.0×10⁶ PBMCs canbe incubated in 10 μg/ml of peptide for 5 hours at 37° C.

Proliferation of the incubated T cells may be measured by any suitablemethod. For example by flow cytometric measurement of incorporation ofthe fluorescent compound CFSE following incubation with peptide, or bymeasuring incorporation of the radiolabelled compound ³H-thymidinefollowing incubation with peptide. A typical example of the lattermethod is as follows:

100 μl of the appropriate peptide concentration is distributed into theappropriate wells of 96 well plates. The plates are then placed into ahumidified 5% CO₂ incubator set at 37° C. for a maximum of 4 hours.PBMC's isolated as standard in the art are prepared to a concentrationof 2×10⁶ cells/ml in complete medium at room temperature. 100 μl of cellsolution is then distributed into each of the wells of the 96 wellplates containing antigen/peptide. The plates are then incubated for 6to 8 days. The cultures are pulsed with tritiated thymidine solution byadding 10 μl of tritiated thymidine stock solution (1.85 MBq/ml inserum-free RPMI medium) to each well. The plates are then returned tothe incubator for between 8 and 16 hours. Cultures are then harvested onto filter mats and dried filter mats are counted using an appropriatebeta scintillation counter. Counts from wells containing peptide arecompared statistically to wells containing media alone (12 wells pergroup). A statistically significant difference between media only wellsand peptide-stimulated wells is considered a positive stimulation ofPBMC's by the peptide or combination of peptides.

Cytokine release may be measured by any suitable method such as ELISAassay as described above. Such methods are well known in the art.

Combination Immunotherapy

Since many individuals are allergic, or may require desensitizing toseveral polypeptide antigens, the current invention also provides meansof desensitizing individuals that are allergic to multiple antigens.“Tolerance” induced in an individual to a first polypeptide antigen orallergen can create in the individual a “tolergeneic environment”wherein inappropriate immune responses to other antigens can bedownregulated in order to provide tolerance to other antigens.

This finding means that individuals allergic to multiple allergens canbe treated in a greatly reduced time period, and that individualsseriously allergic to some allergens (e.g., peanuts) but more mildlyallergic to other allergens (e.g., cat dander) can benefit from atherapy wherein tolerance to the milder allergen is established and thenthis tolergeneic environment is used to provide tolerance to the other,more extreme allergen. In addition, individuals suffering from anautoimmune disorder who are additionally sensitised (or otherwiseimmune) to an unrelated antigen or allergen can benefit from a treatmentregime wherein tolerance to the unrelated antigen or allergen is firstestablished and then this tolergeneic environment is used to providetolerance to the autoantigen associated with the autoimmune disorder.

A method is therefore provided for desensitising a cat allergicindividual to Feld1 antigen and one or more further differentpolypeptide antigens. The method entails, in a first step, administeringto the individual a composition/product/formulation (primarycomposition) according to the invention as described herein and whereinthe administration is carried out in a manner sufficient to generate ahyporesponsive state against the Feld1 antigen. Once a hyporesponsivestate has been established toward Feld1 antigen, or at least a shifttoward desensitisation has occurred, the method entails administrationof a secondary composition comprising a second, different polypeptideantigen to which the individual is to be sensitised. Administration ofthe secondary composition is carried out in such a way as to takeadvantage of the tolergeneic environment established by use of theprimary composition, where it is now possible to establish tolerance tothe second, different polypeptide antigen. The secondary composition iscoadministered with either the first primary composition or a largerfragment of Feld1. By “coadministered” it is meant either thesimultaneous or concurrent administration, e.g., when the two arepresent in the same composition or administered in separate compositionsat nearly the same time but at different sites, as well as the deliveryof polypeptide antigens in separate compositions at different times. Forexample, the secondary composition may be delivered prior to orsubsequent to delivery of the first composition (or a larger fragment ofFeld1) at the same or a different site. The timing between deliveriescan range from about several seconds apart to about several minutesapart, several hours apart, or even several days apart. Furthermore,different delivery methods can be employed.

The second polypeptide antigen is preferably an allergen different toFeld1 allergen. Suitable allergens for use in the methods of theinvention can of course be obtained and/or produced using known methods.Classes of suitable allergens include, but are not limited to, pollens,animal dander other than cat dander, grasses, molds, dusts, antibiotics,stinging insect venoms, and a variety of environmental (includingchemicals and metals), drug and food allergens. Common tree allergensinclude pollens from cottonwood, popular, ash, birch, maple, oak, elm,hickory, and pecan trees; common plant allergens include those frommugwort, ragweed, English plantain, sorrel-dock and pigweed; plantcontact allergens include those from poison oak, poison ivy and nettles;common grass allergens include rye grass, Timothy, Johnson, Bermuda,fescue and bluegrass allergens; common allergens can also be obtainedfrom molds or fungi such as Alternaria, Fusarium, Hormodendrum,Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes;epidermal allergens can be obtained from house or organic dusts(typically fungal in origin), from arthropods such as house mites(Dermatophagoides pteronyssinus), or from animal sources such asfeathers, and dog dander; common food allergens include milk and cheese(diary), egg, wheat, nut (e.g., peanut), seafood (e.g., shellfish), pea,bean and gluten allergens; common environmental allergens include metals(nickel and gold), chemicals (formaldehyde, trinitrophenol andturpentine), Latex, rubber, fiber (cotton or wool), burlap, hair dye,cosmetic, detergent and perfume allergens; common drug allergens includelocal anesthetic and salicylate allergens; antibiotic allergens includepenicillin, tetracycline and sulfonamide allergens; and common insectallergens include bee, wasp and ant venom, and cockroach calyxallergens. Particularly well characterized allergens include, but arenot limited to, the major and cryptic epitopes of the Der p I allergen(Hoyne et al. (1994) Immunology 83190-195), bee venom phospholipase A2(PLA) (Akdis et al. (1996) J. Clin. Invest. 98:1676-1683), birch pollenallergen Bet v 1 (Bauer et al. (1997) Clin. Exp. Immunol. 107:536-541),and the multi-epitopic recombinant grass allergen rKBG8.3 (Cao et al.(1997) Immunology 90:46-51). These and other suitable allergens arecommercially available and/or can be readily prepared as extractsfollowing known techniques.

Preferably, the second polypeptide allergen is selected from the list ofallergen sequences and database accession numbers (NCBI Entrez accessionnumbers) below. NCBI is the National Center for Biotechnologyinformation and is a division of the US National Institutes of Health.The NCBI web site, from which access to the database may be sought, iswww.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers(NCBI Entrez accession numbers):

House Dust Mite

Dermatophagoides pteronyssinus

Der p 1 MKIVLAIASLLALSAVYARPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYG YFAANIDLMMIEEYPYVVILDer p 2 MMYKILCLSLLVAAVARDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFEANQNTKTAKIEIKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRD Der p 3MIIYNILIVLLLAINTLANPILPASPNATIVGGEKALAGECPYQISLQSSSHFCGGTILDEYWILTAAHCVAGQTASKLSIRYNSLKHSLGGEKISVAKIFAHEKYDSYQIDNDIALIKLKSPMKLNQKNAKAVGLPAKGSDVKVGDQVRVSGWGYLEEGSYSLPSELRRVDIAVVSRKECNELYSKANAEVTDNMICGGDVANGGKDSCQGDSGGPVVDVKNNQVVGIVSWGYGCARKGYPGVYTRVGN FIDWIESKRSQ Der p 4KYXNPHFIGXRSVITXLME Der p 5MKFIIAFFVATLAVMTVSGEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQEQINHFEEKPTKEMKDKIVAEMDTIIAMIDGVRGVLDRLMQRKDLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV Der p 6 AIGXQPAAEAEAPFQISLMK Der p 7MMKLLLIAAAAFVAVSADPIHYDKITEEINKAVDEAVAAIEKSETFDPMKVPDHSDKFERHIGIIDLKGELDMRNIQVRGLKQMKRVGDANVKSEDGVVKAHLLVGVHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVELSLEVSEEGNMTLTSFEVRQFANVVNHIGGLSILDPIFAVLSDVLTAIFQDTVRAEMTK VLAPAFKKELERNNQ Derp9 IVGGSNASPGDAVYQIALDermatophagoides farinae

Der f 1 MKFVLAIASLLVLTVYARPASIKTFEFKKAFNKNYATVEEEEVARKNFLESLKYVEANKGAINHLSDLSLDEFKNRYLMSAEAFEQLKTQFDLNAETSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSEQELVDCASQHGCHGDTIPRGIEYIQQNGVVEERSYPYVAREQRCRRPNSQHYGISNYCQIYPPDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGYQPNYHAVNIVGYGSTQGDDYWIVRNSWDTTWGDSGYGY FQAGNNLMMIEQYPYVVIMDer f 2 MISKILCLSLLVAAVVADQVDVKDCANNEIKKVMVDGCHGSDPCIIHRGKPFTLEALFDANQNTKTAKIEIKASLDGLEIDVPGIDTNACHFMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKLIGDNGVLACAIATHGKIRD Der f 3MMILTIVVLLAANILATPILPSSPNATIVGGVKAQAGDCPYQISLQSSSHFCGGSILDEYWILTAAHCVNGQSAKKLSIRYNTLKHASGGEKIQVAEIYQHENYDSMTIDNDVALIKLKTPMTLDQTNAKPVPLPAQGSDVKVGDKIRVSGWGYLQEGSYSLPSELQRVDIDVVSREQCDQLYSKAGADVSENMICGGDVANGGVDSCQGDSGGPVVDVATKQIVGIVSWGYGCARKGYPGVYTRVGNFV DWIESKRSQ Der f 4AVGGQDADLAEAPFQISLLK Der f 7MMKFLLIAAVAFVAVSADPIHYDKITEEINKAIDDAIAAIEQSETIDPMKVPDHADKFERHVGIVDFKGELAMRNIEARGLKQMKRQGDANVKGEEGIVKAHLLIGVHDDIVSMEYDLAYKLGDLHPTTHVISDIQDFVVALSLEISDEGNITMTSFEVRQFANVVNHIGGLSILDPIFGVLSDVLTAIFQDTVRKEMTK VLAPAFKRELEKNAdditional mite allergen sequences (NCBI entrez accession):1170095; 1359436; 2440053; 666007; 487661; 1545803; 84702; 84699;625532; 404370; 1091577; 1460058; 7413; 9072; 387592.

Cat

Felis sequences (NCBI entrez accession):539716; 539715; 423193; 423192; 423191; 423190; 1364213; 1364212;395407; 163827; 163823; 163825; 1169665; 232086; 1169666.

Latex

Hevea sequences:

Hev b 1 MAEDEDNQQGQGEGLKYLGFVQDAATYAVTTFSNVYLFAKDKSGPLQPGVDIIEGPVKNVAVPLYNRFSYIPNGALKFVDSTVVASVTIIDRSLPPIVKDASIQVVSAIRAAPEAARSLASSLPGQTKILAKVFYGEN Hev b 3MAEEVEEERLKYLDFVRAAGVYAVDSFSTLYLYAKDISGPLKPGVDTIENVVKTVVTPVYYIPLEAVKFVDKTVDVSVTSLDGVVPPVIKQVSAQTYSVAQDAPRIVLDVASSVFNTGVQEGAKALYANLEPKAEQYAVITWRALNKLPLVPQVANVVVPTAVYFSEKYNDVVRGTTEQGYRVSSYLPLLPTEKITKVFG DEASAdditional Hevea sequences (NCBI entrez accession):3319923; 3319921; 3087805; 1493836; 1480457; 1223884; 3452147; 3451147;1916805; 232267; 123335; 2501578; 3319662; 3288200; 1942537; 2392631;2392630; 1421554; 1311006; 494093; 3183706; 3172534; 283243; 1170248;1708278; 1706547; 464775; 266892; 231586; 123337; 116359; 123062;2213877; 542013; 2144920; 1070656; 2129914; 2129913; 2129912; 100135;82026; 1076559; 82028; 82027; 282933; 280399; 100138; 1086972; 108697;1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 913758; 913757;913756; 234388; 1092500; 228691; 1177405; 18839; 18837; 18835; 18833;18831; 1209317; 1184668; 168217; 168215; 168213; 168211; 168209; 348137.

Rye Grass

Lolium sequences:

126385 Lol p 1 MASSSSVLLVVALFAVFLGSAHGIAKVPPGPNITAEYGDKWLDAKSTWYGKPTGAGPKDNGGACGYKNVDKAPFNGMTGCGNTPIFKDGRGCGSCFEIKCTKPESCSGEAVTVTITDDNEEPIAPYHFDLSGHAFGSMAKKGEEQNVRSAGELELQFRRVKCKYPDDTKPTFHVEKASNPNYLAILVKYVDGDGDVVAVDIKEKGKDKWIELKESWGAVWRIDTPDKLTGPFTVRYTTEGGTKSEFEDVI PEGWKADTSYSAK 126386Lol p 2a AAPVEFTVEKGSDEKNLALSIKYNKEGDSMAEVELKEHGSNEWLALKKNGDGVWEIKSDKPLKGPFNFRFVSEKGMRNVFDDVVPADFKVGTTYKPE 126387 Lol p 3TKVDLTVEKGSDAKTLVLNIKYTRPGDTLAEVELRQHGSEEWEPMTKKGNLWEVKSAKPLTGPMNFRFLSKGGMKNVFDEVIPTAFTVGKTYTPEYN 2498581 Lol p 5aMAVQKYTVALFLRRGPRGGPGRSYAADAGYTPAAAATPATPAATPAGGWREGDDRRAEAAGGRQRLASRQPWPPLPTPLRRTSSRSSRPPSPSPPRASSPTSAAKAPGLIPKLDTAYDVAYKAAEAHPRGQVRRLRHCPHRSLRVIAGALEVHAVKPATEEVLAAKIPTGELQIVDKIDAAFKIAATAANAAPTNDKFTVFESAFNKALNECTGGAMRPTSSSPPSRPRSSRPTPPPSPAAPEVKYAVFEAALTKAITAMTQAQKAGKPAAAAATAAATVATAAATAAAVLPPPLLVVQS LISLLIYY 2498582 Lolp 5b MAVQKHTVALFLAVALVAGPAASYAADAGYAPATPATPAAPATAATPATPATPATPAAVPSGKATTEEQKLIEKINAGFKAAVAAAAVVPPADKYKTFVETFGTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDAYVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTAATAANAAPANDKFTVFENTFNNAIKVSLGAAYDSYKFIPTLVAAVKQAYAAKQATAPEVKYTVSETALKKAVTAMSEAEKEATPAAAATATPTPAAATATATPAAAYATATPAAATATATPAAATATPAAAGGYKV 455288 Lol p isoform 9MAVQKHTVALFLAVALVAGPAASYAADAGYAPATPATPAAPATAATPATPATPATPAAVPSGKATTEEQKLIEKINAGFKAAVAAAAVVPPADKYKTFVETFGTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDAYVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTAATAANAAPANDKFTVFENTFNNAIKVSLGAAYDSYKFIPTLVAAVKQAYAAKQATAPEVKYTVSETALKKAVTAMSEAEKEATPAAAATATPTPAAATATATPAAAYATATPAAATATATPAAATATPAAAGGYKV 1582249 Lol p 11DKGPGFVVTGRVYCDPCRAGFETNVSHNVEGATVAVDCRPFDGGESKLKAEATTDKDGWYKIEIDQDHQEEICEVVLAKSPDKSCSEIEEFRDRARVPLTSNXGIKQQGIRYANPIAFFRKEPLKECGGILQAYAdditional Lolium sequences (NCBI entrez accession):135480; 417103; 687261; 687259; 1771355; 2388662; 631955; 542131;542130; 542129; 100636; 626029; 542132; 320616; 320615; 320614; 100638;100634; 82450; 626028; 100639; 283345; 542133; 1771353; 1763163;1040877; 1040875; 250525; 551047; 515377; 510911; 939932; 439950; 2718;168316; 168314; 485371; 2388664; 2832717; 2828273; 548867.

Olive Tree

Olive sequences

416610 Ole e 1 EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLRLQCKDKENGDVTFTEVGYTRAEGLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPPNM

Parietaria

Parietaria sequences:

2497750 Par j P2 MRTVSMAALVVIAAALAWTSSAEPAPAPAPGEEACGKVVQDIMPCLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKNELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY 1352506 Par j P5MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRHGPVTGPSDPAHKARLERPQIRVPPPAPEKA 1532056 Par j P8MRTVSMAALVVIAAALAWTSSAELASAPAPGEGPCGKVVHHIMPCLKFVKGEEKEPSKSCCSGTKKLSEEVKTTEQKREACKCIVAATKGISGIKNELVAEVPKKCGITTTLPPITADFDCSKIESTIFRGYY 1532058 Par j P9MRTVSAPSAVALVVIVAAGLAWTSLASVAPPAPAPGSEETCGTVVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGLQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTLGVVPRQPQLPVSLRHGPVTGPSDPAHKARLERPQIRVPPPAPEKA 2497749 Par j P9MRTVSARSSVALVVIVAAVLVWTSSASVAPAPAPGSEETCGTVVGALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTLGVLHYKGN 1086003 Par j 1MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRHGPVTGPSRSRPPTKHGWRDPRLEFRPPHRKKPNPAFSTLGAdditional Parietaria sequences (NCBI entrez accession):543659; 1836011; 1836010; 1311513; 1311512; 1311511; 1311510; 1311509;240971.

Timothy Grass

Phleum sequences:

Phl p 1 MASSSSVLLVVVLFAVFLGSAYGIPKVPPGPNITATYGDKWLDAKSTWYGKPTGAGPKDNGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGHAFGAMAKKGDEQKLRSAGELELQFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKYVNGDGDVVAVDIKEKGKDKWIELKESWGAIWRIDTPDKLTGPFTVRYTTEGGIKTEAEDVI PEGWKADTSYESK Phl p 1MASSSSVLLVVALFAVFLGSAHGIPKVPPGPNITATYGDKWLDAKSTWYGKPTAAGPKDNGGACGYICDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAAYHFDLSGIAFGSMAKKGDEQKLRSAGEVEIQFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKFSGDGDVVAVDIKEKGKDKWIALKESWGAIWRIDTPEVLKGPFTVRYTTEGGTKARAKDVI PEGWKADTAYESK Phlp 2MSMASSSSSSLLAMAVLAALFAGAWCVPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELREHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE Phl p 5ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p 5ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p 5bAAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5aADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATAATGGYKV Phl p 5MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVSYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGICATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEDPAWPKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTATGAASGAATVAAGGYKV Phl p 5ADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAG AASGAATVAAGGYKVPhl p 5 SVKRSNGSAEVHRGAVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5MAVHQYTVALFLAVALVAGPAGSYAADLGYGPATPAAPAAGYTPATPAAPAGAEPAGKATTEEQKLIEKINAGFKAALAAAAGVPPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATVSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 5ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p5bAAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p5aADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATAATGGYKV Phl p5AVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAGDKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTATGAASGAATVAAGGYKV Phl p 5bMAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAARQRPAADKFKTFEAASPRHPRPLRQGAGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAENDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 5EAPAGKATTEEQKLIEKINAGFKAALARRLQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAAELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPPPLPPPPQPPPLAATGAATA ATGGYKV Phl p 5MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVIDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAA TGAATAATGGYKVPhl p 5b MAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAARQRPAADKFKTFEAASPRHPRPLRQGAGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5aADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPPPLPPPPQPPPLAATGAATAATGGYKV Phl p 5MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 6MAAHKFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA Phl p 6SKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEV HAVKPGA Phl p 6ADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEVHAVKPGA Phl p 6TEEQKLIEDVNASFRAAMATTANVPPADKYKTLEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVH AVKPGA Phl p 6MAAHKFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDINASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEVHAVKPGA Phl p6MVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA Phl p 7MADDMERIFKRFDTNGDGKISLSELTDALRTLGSTSADEVQRMMAEIDTDGDGFIDFNEFISFCNANPGLMKDVAKVF Phl p 11MSWQTYVDEHLMCEIEGHHLASAAILGHDGTVWAQSADFPQFKPEEITGIMKDFDEPGHLAPTGMFVAGAKYMVIQGEPGRVIRGKKGAGGITIKKTGQALVVGIYDEPMTPGQCNMVVERLGDYLVEQGMAdditional Phleum sequences (NCBI entrez accession):458878; 548863; 2529314; 2529308; 2415702; 2415700; 2415698; 542168;542167; 626037; 542169; 541814; 542171; 253337; 253336; 453976; 439960.

Wasp (and Related)

Vespula sequences:

465054 ALLERGEN VES V 5MEISGLVYLIIIVTIIDLPYGKANNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQDLLKEHNDFRQKIARGLETRGNPGPQPPAKNMKNLVWNDELAYVAQVWANQCQYGHDTCRDVAKYQVGQNVALTGSTAAKYDDPVKLVKMWEDEVKDYNPKKKFSGNDFLKTGHYTQMVWANTKEVGCGSIKYIQEKWHKHYLVCNYGPSGNFMNEELYQTK 1709545 ALLERGEN VES M 1GPKCPFNSDTVSIIIETRENRNRDLYTLQTLQNHPEFKKKTITRPVVFITHGFTSSASEKNFINLAKALVDKDNYMVISIDWQTAACTNEYPGLKYAYYPTAASNTRLVGQYIATITQKLVKDYKISMANIRLIGHSLGAHVSGFAGKRVQELKLGKYSEIIGLDPARPSFDSNHCSERLCETDAEYVQIIHTSNYLGTEKILGTVDFYMNNGKNNPGCGRFFSEVCSHTRAVIYMAECIKHECCLIGIPRSKSSQPISRCTKQECVCVGLNAKKYPSRGSFYVPVESTAPFCNNKGKII1352699 ALLERGEN VES V 1MEENMNLKYLLLFVYFVQVLNCCYGHGDPLSYELDRGPKCPFNSDTVSIIIETRENRNRDLYTLQTLQNHPEFKKKTTIRPVVFITHGFTSSASETNFINLAKALVDKDNYMVISIDWQTAACTNEAAGLKYLYYPTAARNTRLVGQYIATITQKLVKHYKISMANIRLIGHSLGAHASGFAGKKVQELKLGKYSEIIGLDPARPSFDSNHCSERLCETDAEYVQIIHTSNYLGTEKTLGTVDFYMNNGKNQPGCGRFFSEVCSHSRAVIYMAECIKHECCLIGIPKSKSSQPISSCTKQECVCVGLNAKKYPSRGSFYVPVESTAPFCNNKGKII 1346323 ALLERGEN VES V 2SERPKRVFNIYWNVPTFMCHQYDLYFDEVTNFNIKRNSKDDFQGDKIALFYDPGEFPALLSLKDGKYKKRNGGVPQEGNITIHLQKFIENLDKIYPNRNFSGIGVIDFERWRPIFRQNWGNMKIHKNFSIDLVRNEHPTWNKKMIELEASKRFEKYARFFMEETLKLAKKTRKQADWGYYGYPYCFNMSPNNLVPECDVTAMHENDKMSWLFNNQNVLLPSVYVRQELTPDQRIGLVQGRVKEAVRISNNLKHSPKVLSYWWYVYQDETNTFLTETDVKKTFQEIVINGGDGIIIWGSSSDVNSLSKCKRLQDYLLTVLGPIAINVTEAVN 549194 ALLERGEN VES VI5KVNYCKIKCLKGGVHTACKYGTSTKPNCGKMVVKAYGLTEAEKQEILKVHNDFRQKVAKGLETRGNPGPQPPAKNMNNLVWNDELANIAQVWASQCNYGHDTCKDTEKYPVGQNIAKRSTTAALFDSPGKLVKMWENEVKDFNPNIEWSKNNLKKTGHYTQMVWAKTKEIGCGSVKYVKDEWYTHYLVCNYGPSGNFRN EKLYEKKAdditional vespula sequences (NCBI entrez accession):549193; 549192; 549191; 549190; 549189; 117414; 126761; 69576; 625255;627189; 627188; 627187; 482382; 112561; 627186; 627185; 1923233; 897645;897647; 745570; 225764; 162551.

Tree Allergen Sequences (Mainly Birch) Sequences:

114922 Bet v 1 MGVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVES YLLAHSDAYN130975 Bet v 2 MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 1168696 Bet v 3MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLFDKNSDGITTVDELSRALNLLGLETDLSELESTVKSFTREGNIGLQFEDFISLHQSLNDSYFAYGGEDEDDNEEDMRKSILSQEEADSFGGFKVFDEDGDGYISARELQMVLGKLGFSEGSEIDRVEKMIVSVDSNRDGRVDFFEFKDMMRSV LVRSS 809536 Bet v 4MADDHPQDKAERERIFKRFDANGDGKISAAELGEALKTLGSITPDEVKHMMAEIDTDGDGFISFQEFTDFGRANRGLLKDVAKIF 543675 Que a I - Quercus alba =oak trees (fragment) GVFTXESQETSVIAPAXLFKALFL543509 Car b I - Carpinus betulus = hornbeam trees (fragment)GVFNYEAETPSVIPAARLFKSYVLDGDKLIPKVAPQAIXK543491 Aln g I - Alnus glutinosa = alder trees (fragment)GVFNYEAETPSVIPAARLFKAFILDGDKLLPKVAPEAVSSVENI 1204056 RubiscoVQCMQVWPPLGLKKFETLSYLPPLSSEQLAKEVDYLLRKNLIPCLEFELEHGFVYREHNRSPGYYDGRYWTMWKLPMFGCNDSSQVLKELEECKKAYPSA FIRIIGFDDKAdditional tree allergen sequences (NCBI entrez accession number):131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728;1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712;3015520; 2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708;1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1842188; 2564228;2564226; 2564224; 2564222; 2564220; 2051993; 1813891; 1536889; 534910;534900; 534898; 1340000; 1339998; 2149808; 66207; 2129477; 1076249;1076247; 629480; 481805; 81443; 1361968; 1361967; 1361966; 1361965;1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 629483;629482; 629481; 541804; 320545; 81444; 541814; 629484; 474911; 452742;1834387; 298737; 298736; 1584322; 1584321; 584320; 1542873; 1542871;1542869; 1542867; 1542865; 1542863; 1542861; 1542859; 1542857; 1483232;1483230; 1483228; 558561; 551640; 488605; 452746; 452744; 452740;452738; 452736; 452734; 452732; 452730; 452728; 450885; 17938; 17927;17925; 17921; 297538; 510951; 289331; 289329; 166953.

Peanut

Peanut sequences

1168391 Ara h 1 MRGRVSPLMLLLGILVLASVSATHAKSSPYQKKTENPCAQRCLQSCQQEPDDLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDDDRRQPRREEGGRWGPAGPREREREEDWRQPREDWRRPSHQQPRKIRPEGREGEQEWGTPGSHVREETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSRQFQNLQNHRIVQIEAKPNTLVLPKHADADNILVIQQGQATVTVANGNNRKSFNLDEGHALRIPSGFISYILNRHDNQNLRVAKISMPVNTPGQFEDEFPASSRDQSSYLQGFSRNTLEAAFNAEFNEIRRVLLEENAGGEQEERGQRRWSTRSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDLSNNFGKLFEVKPDKKNPQLQDLDMMLTCVEIKEGALMLPHFNSKAMVIVVVNKGTGNLELVAVRKEQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPVAINASSELHLLGFGINAENNHRIFLAGDKDNVIDQIEKQAKDLAFPGSGEQVEKLIKNQKESHFVSARPQSQSQSPSSPEKESPEKEDQEEENQGGKGPLLSILKAFN

Ragweed

Ambrosia sequences

113478 Amb a 1 MGIKHCCYILYFTLALVTLLQPVRSAEDLQQILPSANETRSLTTCGTYNIIDGCWRGKADWAENRKALADCAQGFAKGTIGGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWIIFARDMVIRLDRELAINNDKTIDGRGAKVEIINAGFAIYNVKNIIIHNIIMHDIVVNPGGLIKSHDGPPVPRKGSDGDAIGISGGSQIWIDHCSLSKAVDGLIDAKHGSTHFTVSNCLFTQHQYLLLFWDFDERGMLCTVAFNKFTDNVDQRMPNLRHGFVQVVNNNYERWGSYALGGSAGPTILSQGNRFLASDIKKEVVGRYGESAMSESINWNWRSYMDVFENGAIFVPSGVDPVLTPEQNAGMIPAEPGEAVLRLTSSAGVLSCQPGAPC 113479 Amb a 2MGIKHCCYILYFTLALVTLVQAGRLGEEVDILPSPNDTRRSLQGCEAHNIIDKCWRCKPDWAENRQALGNCAQGFGKATHGGKWGDIYMVTSDQDDDVVNPKEGTLRFGATQDRPLWIIFQRDMIIYLQQEMVVTSDKTIDGRGAKVELVYGGITLMNVKNVIIHNIDIHDVRVLPGGRIKSNGGPAIPRHQSDGDAIHVTGSSDIWIDHCTLSKSFDGLVDVNWGSTGVTISNCKFTHHEKAVLLGASDTHFQDLKMHVTLAYNIFTNTVHERMPRCRFGFFQIVNNFYDRWDKYAIGGSSNPTILSQGNKFVAPDFIYKKNVCLRTGAQEPEWMTWNWRTQNDVLENGAIFVASGSDPVLTAEQNAGMMQAEPGDMVPQLTMNAGVLTCSPGAPC 113477 Amb a 1.3MGIKQCCYILYFTLALVALLQPVRSAEGVGEILPSVNETRSLQACEALNIIDKCWRGKADWENNRQALADCAQGFAKGTYGGKWGDVYTVTSNLDDDVANPKEGTLRFAAAQNRPLWIIFKNDMVINLNQELVVNSDKTIDGRGVKVEIINGGLTLMNVKNIIIHNINIHDVKVLPGGMIKSNDGPPILRQASDGDTINVAGSSQIWIDHCSLSKSFDGLVDVTLGSTHVTISNCKFTQQSKAILLGADDTIIVQDKGMLATVAFNMFTDNVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGSSAPTILCQGNRFLAPDDQIKKNVLARTGTGAAESMAWNWRSDKDLLENGAIFVTSGSDPVLTPVQSAGMIPAEPGEAAIKLTSSAGVFSCHPGAPC 113476 Amb a 1.2MGIKHCCYILYFTLALVTLLQPVRSAEDVEEFLPSANETRRSLKACEAHNIIDKCWRCKADWANNRQALADCAQGFAKGTYGGKHGDVYTVTSDKDDDVANPKEGTLRFAAAQNRPLWIIFKRNMVIHLNQELVVNSDKTIDGRGVKVNIVNAGLTLMNVKNIIIHNINIHDIKVCPGGMIKSNDGPPILRQQSDGDAINVAGSSQIWIDHCSLSKASDGLLDITLGSSHVTVSNCKFTQHQFVLLLGADDTHYQDKGMLATVAFNMFTDHVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGSSAPTILSQGNRFFAPDDIIKKNVLARTGTGNAESMSWNWRTDRDLLENGAIFLPSGSDPVLTPEQKAGMIPAEPGEAVLRLTSSAGVLSCHQGAPC 113475 Amb a 1.1MGIKHCCYILYFTLALVTLLQPVRSAEDLQEILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALADCAQGFGKGTVGGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWIIFERDMVIRLDKEMVVNSDKTIDGRGAKVEIINAGFTLNGVKNVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAISISGSSQIWIDHCSLSKSVDGLVDAKLGTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVAFNTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGSASPTILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRTNKDVLENGAIFVASGVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPGAPC

Cedar Sequences

493634 Cry j IB precursorMDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTTYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLSKRC 493632 Cry j IA precursorMDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTTYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCSLSKRC 1076242 Cry j II precursor - Japanese cedarMAMKLIAPMAFLAMQLIIMAAAEDQSAQIMLDSVVEKYLRSNRSLRKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKNPSAMLLVPGSKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVENMRAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYP QRWICSCHGKIYHP1076241 Cry j II protein - Japanese cedarMAMKFIAPMAFVAMQLIIMAAAEDQSAQIMLDSDIEQYLRSNRSLRKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKKPSAMLLVPGNKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVKNMGAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYP QRWMCSRHGKIYHP541803 Cry j I precursor - Japanese cedarMDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTTYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCSLSKRC 541802 Cry j I precursor- Japanese cedarMDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLSKRC

Dog

Canis sequences:

Can f 1 MKTLLLTIGFSLIAILQAQDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGGQ Serum albumin fragment EAYKSEIAHRYNDLGEEHFRGLVLSerum albumin fragmentLSSAKERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVHKECCHGDLLECADDRADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVVEVSRKLGKVGTKCCK KPESERMSCADDFLSCan f 2 MQLLLLTVGLALICGLQAQEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLSRQQDFLPAFESVCEDIGLHKDQIVVLSDDDRCQGSRDAdditional dog allergen protein (NCBI entrez accession):1731859

Horse

Equus sequences:

1575778 Equ cl MKLLLLCLGLILVCAQQEENSDVAIRNFDISKISGEWYSIFLASDVKEKIEENGSMRVFVDVIRALDNSSLYAEYQTKVNGECTEFPMVFDKTEEDGVYSLNYDGYNVFRISEFENDEHIILYLVNFDKDRPFQLFEFYAREPDVSPEIKEEFVKIVQKRGIVKENIIDLTKIDRCFQLRGNGVAQA 3121755 Equ c 2SQXPQSETDYSQLSGEWNTIYGAASNIXK

Euroglyphus (Mite)

Euroglyphus sequences:

Eur m 1 (variant) TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYRNMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQSCHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYDGRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSWDTTWGDN GYGYFAANINLEur m 1 (variant) TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYRNMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQSCHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYDGRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSWDTTWGDN GYGYFAANINLEur m 1 (variant) ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNANKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDN GYGYFAANIDLEur m 1 (variant) ETSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSEQELVDCASQHGCHGDTIPRGIEYIQQNGVVEERSYPYVAREQQCRRPNSQHYGISNYCQIYPPDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGYQPNYHAVNIVGYGSTQGVDYWIVRNSWDTTWGD SGYGYFQAGNNL

Poa (Grass) Sequences

113562 POLLEN ALLERGEN POA P 9MAVQKYTVALFLVALVVGPAASYAADLSYGAPATPAAPAAGYTPAAPAGAAPKATTDEQKMIEKINVGFKAAVAAAGGVPAANKYKTEVATFGAASNKAFAEALSTEPKGAAVDSSKAALTSKLDAAYKLAYKSAEGATPEAKYDDYVATLSEALRIIAGTLEVHGVKPAAEEVKATPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAATGTATAAVGAATGAATA AAGGYKV113561 POA P 9 MAVHQYTVALFLAVALVAGPAASYAADVGYGAPATLATPATPAAPAAGYTPAAPAGAAPKATTDEQKLIEKINAGFKAAVAAAAGVPAVDKYKTFVATFGTASNKAFAEALSTEPKGAAAASSNAVLTSKLDAAYKLAYKSAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAGEEVKAIPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAVTATATGAVGAATGAVGAATGAATAAAGGYKTGAATPTAGGYKV 113560 POA P 9MDKANGAYKTALKAASAVAPAEKFPVFQATFDKNLKEGLSGPDAVGFAKKLDAFIQTSYLSTKAAEPKEKFDLFVLSLTEVLRFMAGAVKAPPASKEPAKPAPKVAAYTPAAPAGAAPKATTDEQKLIEKINVGFKAAVAAAAGVPAASKYKTFVATFGAASNKAFAEALSTEPKGAAVASSKAVLTSKLDAAYKLAYKSAEGATPEAKYDAYVATLSEALRIIAGTLEVHGVKPAAEEVKAIPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAVTGTATSAVGAATGAATAAAGGYKV

Cockroach Sequences

2833325 Cr p1 MKTALVFAAVVAFVAARFPDHKDYKQLADKQFLAKQRDVLRLFHRVHQHNILNDQVEVGIPMTSKQTSATTVPPSGEAVHGVLQEGHARPRGEPFSVNYEKHREQAIMLYDLLYFANDYDTFYKTACWARDRVNEGMFMYSFSIAVFHRDDMQGVMLPPPYEVYPYLFVDHDVIHMAQKYWMKNAGSGEHHSHVIPVNFTLRTQDHLLAYFTSDVNLNAFNTYYRYYYPSWYNTTLYGHNIDRRGEQFYYTYKQIYARYFLERLSNDLPDVYPFYYSKPVKSAYNPNLRYHNGEEMPVRPSNMYVTNFDLYYIADIKNYEKRVEDAIDFGYAFDEHMKPHSLYHDVHGMEYLADMIEGNMDSPNFYFYGSIYHMYHSMIGHIVDPYHKMGLAPSLEHPETVLRDPVFYQLWKRVDHLFQKYKNRLPRYTHDELAFEGVKVENVDVGKLYTYFEQYDMSLDMAVYVNNVDQISNVDVQLAVRLNHKPFTYNIEVSSDKAQDVYVAVFLGPKYDYLGREYDLNDRRHYFVEMDRFPYHVGAGKTVIERNSHDSNIIAPERDSYRTFYKKVQEAYEGKSQYYVDKGHNYCGYPENLLIPKGKKGGQAYTFYVIVTPYVKQDEHDFEPYNYKAFSYCGVGSERKYPDNKPLGYPFDRKIYSNDFYTPNMYFKDVIIFHKKYDEVGVQGH 2231297 Cr p2INEIHSIIGLPPFVPPSRRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIRSPEFQSIISTLNAMQRSEHHQNLRDKGVDVDHFIQLIRALFGLSRAARNLQDDLNDFLHSLEPISPRHRHGLPRQRRRSARVSAYLHADDFHKIITTIEALPEFANFYNFLKEHGLDVVDYINEIHSIIGLPPFVPPSRRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIRSPEFQSIISTLNAMPEYQELLQNLRDKGVDVDHFIRVDQGTLRTLSSGQRNLQDDLNDFLALIPTDQILAIAMDYLANDAEVQELVAYLQSDDFHKIITTIEALPEFANFYNFLKEHGLDVVDYINEIHSIIGLPPFVPPSQRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIDLRSSRA 1703445 Bla g 2MIGLKLVTVLFAVATITHAAELQRVPLYKLVHVFINTQYAGITKIGNQNFLTVFDSTSCNVVVASQECVGGACVCPNLQKYEKLKPKYISDGNVQVKFFDTGSAVGRGIEDSLTISNLTTSQQDIVLADELSQEVCILSADVVVGIAAPGCPNALKGKTVLENFVEENLIAPVFSIHHARFQDGEHFGEIIFGGSDWKYVDGEFTYVPLVGDDSWKFRLDGVKIGDTTVAPAGTQAIIDTSKAIIVGPKAYVNPINEAIGCVVEKTTTRRICKLDCSKIPSLPDVTFVINGRNFNISSQYYIQQNGNLCYSGFQPCGHSDHFFIGDFFVDHYYSEFNWENKTMGFGRSVE SV 1705483 Bla g 4AVLALCATDTLANEDCFRHESLVPNLDYERFRGSWIIAAGTSEALTQYKCWIDRFSYDDALVSKYTDSQGKNRTTIRGRTKFEGNKFTIDYNDKGKAFSAPYSVLATDYENYAIVEGCPAAANGHVIYVQIRFSVRRFHPKLGDKEMIQHYTLDQVNQHKKAIEEDLKHFNLKYEDLHSTCH 2326190 Bla g 5YKLTYCPVKALGEPIRFLLSYGEKDFEDYRFQEGDWPNLKPSMPFGKTPVLEIDGKQTHQSVAISRYLGKQFGLSGKDDWENLEIDMIVDTISDFRAAIANYHYDADENSKQKKWDPLKKETIPYYTKKFDEVVKANGGYLAAGKLTWADFYFVAILDYLNHMAKEDLVANQPNLKALREKVLGLPAIKAWVAKRPPTDLAdditional cockroach sequences (NCBI Entrez accession numbers):2580504; 1580797; 1580794; 1362590; 544619; 544618; 1531589; 1580792;1166573; 1176397; 2897849.

Allergen (General) Sequences:

NCBI accession numbers2739154; 3719257; 3703107; 3687326; 3643813; 3087805; 1864024; 1493836;1480457; 2598976; 2598974; 1575778; 763532; 746485; 163827; 163823;3080761; 163825; 3608493; 3581965; 2253610; 2231297; 2897849; 3409499;3409498; 3409497; 3409496; 3409495; 3409494; 3409493; 3409492; 3409491;3409490; 3409489; 3409488; 3409487; 3409486; 3409485; 3409484; 3409483;3409482; 3409481; 3409480; 3409479; 3409478; 3409477; 3409476; 3409475;3409474; 3409473; 3409472; 3409471; 3409470; 3409469; 3409468; 3409467;3409466; 3409465; 3409464; 3409463; 3409462; 3409461; 3409460; 3409459;3409458; 3409457; 3409456; 3318885; 3396070; 3367732; 1916805; 3337403;2851457; 2851456; 1351295; 549187; 136467; 1173367; 2499810; 2498582;2498581; 1346478; 1171009; 126608; 114091; 2506771; 1706660; 1169665;1169531; 232086; 416898; 114922; 2497701; 1703232; 1703233; 1703233;1703232; 3287877; 3122132; 3182907; 3121758; 3121756; 3121755; 3121746;3121745; 3319925; 3319923; 3319921; 3319651; 3318789; 3318779; 3309647;3309047; 3309045; 3309043; 3309041; 3309039; 3288200; 3288068; 2924494;3256212; 3256210; 3243234; 3210053; 3210052; 3210051; 3210050; 3210049;3210048; 3210047; 3210046; 3210045; 3210044; 3210043; 3210042; 3210041;3210040; 3210039; 3210038; 3210037; 3210036; 3210035; 3210034; 3210033;3210032; 3210031; 3210030; 3210029; 3210028; 3210027; 3210026; 3210025;3210024; 3210023; 3210022; 3210021; 3210020; 3210019; 3210018; 3210017;3210016; 3210015; 3210014; 3210013; 3210012; 3210011; 3210010; 3210009;3210008; 3210007; 3210006; 3210005; 3210004; 3210003; 3210002; 3210001;3210000; 3209999; 3201547; 2781152; 2392605; 2392604; 2781014; 1942360;2554672; 2392209; 3114481; 3114480; 2981657; 3183706; 3152922; 3135503;3135501; 3135499; 3135497; 2414158; 1321733; 1321731; 1321728; 1321726;1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3095075;3062795; 3062793; 3062791; 2266625; 2266623; 2182106; 3044216; 2154736;3021324; 3004467; 3005841; 3005839; 3004485; 3004473; 3004471; 3004469;3004465; 2440053; 1805730; 2970629; 2959898; 2935527; 2935416; 809536;730091; 585279; 584968; 2498195; 2833325; 2498604; 2498317; 2498299;2493414; 2498586; 2498585; 2498576; 2497749; 2493446; 2493445; 1513216;729944; 2498099; 548449; 465054; 465053; 465052; 548671; 548670; 548660;548658; 548657; 2832430; 232084; 2500822; 2498118; 2498119; 2498119;2498118; 1708296; 1708793; 416607; 416608; 416608; 416607; 2499791;2498580; 2498579; 2498578; 2498577; 2497750; 1705483; 1703445; 1709542;1709545; 1710589; 1352699; 1346568; 1346323; 1346322; 2507248; 11352240;1352239; 1352237; 1352229; 1351935; 1350779; 1346806; 1346804; 1346803;1170095; 1168701; 1352506; 1171011; 1171008; 1171005; 1171004; 1171002;1171001; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704;1168703; 1168702; 1168696; 1168391; 1168390; 1168348; 1173075; 1173074;1173071; 1169290; 1168970; 1168402; 729764; 729320; 729979; 729970;729315; 730050; 730049; 730048; 549194; 549193; 549192; 549191; 549190;549189; 549188; 549185; 549184; 549183; 549182; 549181; 549180; 549179;464471; 585290; 416731; 1169666; 113478; 113479; 113477; 113476; 113475;130975; 119656; 113562; 113561; 113560; 416610; 126387; 126386; 126385;132270; 416611; 416612; 416612; 416611; 730035; 127205; 1352238; 125887;549186; 137395; 730036; 133174; 114090; 131112; 126949; 129293; 124757;129501; 416636; 2801531; 2796177; 2796175; 2677826; 2735118; 2735116;2735114; 2735112; 2735110; 2735108; 2735106; 2735104; 2735102; 2735100;2735098; 2735096; 2707295; 2154730; 2154728; 1684720; 2580504; 2465137;2465135; 2465133; 2465131; 2465129; 2465127; 2564228; 2564226; 2564224;2564222; 2564220; 2051993; 1313972; 1313970; 1313968; 1313966; 2443824;2488684; 2488683; 2488682; 2488681; 2488680; 2488679; 2488678; 2326190;2464905; 2415702; 2415700; 2415698; 2398759; 2398757; 2353266; 2338288;1167836; 414703; 2276458; 1684718; 2293571; 1580797; 1580794; 2245508;2245060; 1261972; 2190552; 1881574; 511953; 1532058; 1532056; 1532054;1359436; 666007; 487661; 217308; 1731859; 217306; 217304; 1545803;1514943; 577696; 516728; 506858; 493634; 493632; 2154734; 2154732;543659; 1086046; 1086045; 2147643; 2147642; 1086003; 1086002; 1086001;543675; 543623; 543509; 543491; 1364099; 2147108; 2147107; 1364001;1085628; 631913; 631912; 631911; 2147092; 477301; 543482; 345521;542131; 542130; 542129; 100636; 2146809; 480443; 2114497; 2144915;72355; 71728; 319828; 1082946; 1082945; 1082944; 539716; 539715; 423193;423192; 423191; 423190; 1079187; 627190; 627189; 627188; 627187; 482382;1362656; 627186; 627185; 627182; 482381; 85299; 85298; 2133756; 2133755;1079186; 627181; 321044; 321043; 112559; 112558; 1362590; 2133564;1085122; 1078971; 627144; 627143; 627142; 627141; 280576; 102835;102834; 102833; 102832; 84703; 84702; 84700; 84699; 84698; 84696;477888; 477505; 102575; 102572; 478272; 2130094; 629813; 629812; 542172;542168; 542167; 481432; 320620; 280414; 626029; 542132; 320615; 320614;100638; 100637; 100635; 82449; 320611; 320610; 280409; 320607; 320606;539051; 539050; 539049; 539048; 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Delivery Methods

Once formulated the compositions of the invention can be delivered to asubject in vivo using a variety of known routes and techniques. Forexample, a composition can be provided as an injectable solution,suspension or emulsion and administered via parenteral, subcutaneous,epidermal, intradermal, intramuscular, intraarterial, intraperitoneal,intravenous injection using a conventional needle and syringe, or usinga liquid jet injection system. Compositions can also be administeredtopically to skin or mucosal tissue, such as nasally, intratracheally,intestinal, rectally or vaginally, or provided as a finely divided spraysuitable for respiratory or pulmonary administration. Other modes ofadministration include oral administration, suppositories, sublingualadministration, and active or passive transdermal delivery techniques.

Where a peptide of the invention is to be administered, it is preferredto administer the peptide to a site in the body where it will have theability to contact suitable antigen presenting cells, and where it, orthey, will have the opportunity to contact T cells of the individual.Where an APC is to be administered, it is preferred to administer theAPC to a site in the body where it will have the ability to contact, andactivate, suitable T cells of the individual.

Delivery Regimes

Administration of the peptides/polynucleotides/cells (such as thecomposition containing a plurality of peptides) may be by any suitablemethod as described above. Suitable amounts of the peptide may bedetermined empirically, but typically are in the range given below. Asingle administration of each peptide may be sufficient to have abeneficial effect for the patient, but it will be appreciated that itmay be beneficial if the peptide is administered more than once, inwhich case typical administration regimes may be, for example, once ortwice a week for 2-4 weeks every 6 months, or once a day for a weekevery four to six months. As will be appreciated, each peptide orpolynucleotide, or combination of peptides and/or polynucleotides may beadministered to a patient singly or in combination.

Dosages for administration will depend upon a number of factorsincluding the nature of the composition, the route of administration andthe schedule and timing of the administration regime. Suitable doses ofa molecule or a combination of molecules of the invention may be in theorder of upto 10 μg, up to 15 μg, up to 20 μg, up to 25 μg, up to 30 μg,up to 35 μg, up to 50 μg, up to 100 μg, up to 500 μg or more peradministration. Suitable doses may be less than 15 μg, but at least 1ng, or at least 2 ng, or at least 5 ng, or at least 50 ng, or least 100ng, or at least 500 ng, or at least 1 μg, or at least 10 μg. For somemolecules or combinations of the invention, the dose used may be higher,for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mgor higher. Such doses may be provided in a liquid formulation, at aconcentration suitable to allow an appropriate volume for administrationby the selected route. It will be understood that the above doses referto total dose in the case of a combination of molecules. For example,“up to 35 μg” refers to a total peptide concentration of up to 35 μg ina composition comprising a combination of more than one peptide.

Kits

The invention also relates to a combination of components describedherein suitable for use in a treatment of the invention which arepackaged in the form of a kit in a container. Such kits may comprise aseries of components to allow for a treatment of the invention. Forexample, a kit may comprise four or more different peptides,polynucleotides and/or cells of the invention, or four or more peptides,polynucleotides or cells of the invention and one or more additionaltherapeutic agents suitable for simultaneous administration, or forsequential or separate administration. The kit may optionally containother suitable reagent(s) or instructions and the like.

The invention is illustrated by the following Examples.

Example 1 Screening of Peptide Mixtures for MHC Binding CharacteristicsBinding Assays Peptides

The following peptides that encompass the sequences of Fel d1 wereinvestigated for their capacity to bind the nine HLA-DR molecules: DR1,DR3, DR4, DR7, DR11, DR13, DR15, B4 and B5.

SEQ ID NO: MLA1 H₂N EICPAVKRDVDLFLTGT COOH Derived from Fel d1 chain 1Related to 1 MLA2 H₂N LFLTGTPDEYVEQVAQY COOH Derived from Fel d1 chain 18 MLA3 H₂N EQVAQYKALPVVLENA COOH Derived from Fel d1 chain 1 2 MLA4 H₂NKALPVVLENARILKNCV COOH Derived from Fel d1 chain 1 3 MLA5 H₂NRILKNCVDAKMTEEDKE COOH Derived from Fel d1 chain 1 4 MLA6 H₂NKMTEEDKENALSLLDK COOH Derived from Fel d1 chain 1 9 MLA7 H₂NKENALSVLDKIYTSPL COOH Derived from Fel d1 chain 1 5 MLA8* H₂NVKMAETCPIFYDVFFA COOH Derived from Fel d1 chain 2 13 MLA9* H₂NCPIFYDVFFAVANGNEL COOH Derived from Fel d1 chain 2 14 MLA10* H₂NGNELLLKLSLTKVNAT COOH Derived from Fel d1 chain 2 15 MLA11 H₂NLTKVNATEPERTAMKK COOH Derived from Fel d1 chain 2 10 MLA12 H₂NTAMKKIQDCYVENGLI COOH Derived from Fel d1 chain 2 6 MLA13* H₂NCYVENGLISRVLDGLV COOH Derived from Fel d1 chain 2 16 MLA14 H₂NSRVLDGLVMTTISSSK COOH Derived from Fel d1 chain 2 7 MLA15 H₂NISSSKDCMGEAVQNTV COOH Derived from Fel d1 chain 2 11 MLA16 H₂NAVQNTVEDLKLNTLGR COOH Derived from Fel d1 chain 2 12 *Peptides shown initalics were assessed for binding but not considered further in theseexperiments due to relatively poor solubility.

Binding Conditions for MHC Binding Assays

EBV homozygous cell lines were used as sources of human HLA class IImolecules (Tab. 4). HLA-DR molecules were purified by affinitychromatography using the monomorphic Mab L243 (ATCC, Rockville, USA)coupled to protein A sepharose CL 4B gel (Pharmacia, France). Briefly,cells were lysed on ice at 5×108 cells/ml in 150 mM NaCl, 10 mM Tris HClpH=8.3 buffer containing 1% Nonidet P40 (NP40), 10 mg/l aprotinin, 5 mMEDTA and 10 mM PMSF. After centrifugation at 100 000 g for 1 h, thesupernatant was applied to a sepharose 4B and protein A-sepharose 4Bcolumns and then to the specific affinity column. HLA-DR molecules wereeluted with 1.1 mM n-dodecyl b-D-maltoside (DM), 500 mM NaCl and 500 mMNa2CO3 pH=11.5. Fractions were immediately neutralized to pH=7 with 2 MTris HCl pH=6.8 buffer and extensively dialysed against 1 mM DM, 150 mMNaCl, 10 mM phosphate pH=7 buffer. For HLA-DR molecules beyond lotnumber 40 the 1 mM DM in dialysis buffer was replaced by 1 mM NOGP.

HLA-DR molecules were diluted in 10 mM phosphate, 150 mM NaCl, 1 mM DM,10 mM citrate, 0.003% thimerosal buffer with an appropriate biotinylatedpeptide and serial dilutions of competitor peptides. Binding conditionsof each molecule are detailed in Tab 4. Samples (100 μl per well) wereincubated in 96-wells polypropylene plates (Nunc, Denmark) at 37° C. for24 h to 72 h. After neutralization with 50 μl of 450 mM Tris HCl pH=7.5,0.003% thimerosal, 0.3% BSA, 1 mM DM buffer, samples were applied to96-well maxisorp ELISA plates (Nunc, Denmark) previously coated with 10mg/ml L243 Mab and saturated with 100 mM Tris HCl pH=7.5, 0.3% BSA,0.003% thimerosal buffer. They were allowed to bind to theantibody-coated plates for 2 h at room temperature. Bound biotinylatedpeptide was detected by incubating streptavidine-alkaline phosphataseconjugate (Amersham, U.K.), and after washings, by adding4-methylumbelliferyl phosphate substrate (Sigma, France). Emittedfluorescence was measured at 450 nm upon excitation at 365 nm on aWallac Victor2 1420 multilabel counter fluorimeter (Perkin Elmer).Maximal binding was determined by incubating the biotinylated peptidewith the MHC II molecule in the absence of competitor. Bindingspecificity was assessed by adding an excess of non biotinylatedpeptide. Background did not significantly differ from that obtained byincubating the biotinylated peptide without MHC II molecules. Data wereexpressed as the peptide concentration that prevented binding of 50% ofthe labeled peptide (IC50). Binding ability was then evaluated relativeto known strong binding control (reference) peptide. Suitable referencepeptides for the HLA alleles tested in these experiments are: DR1(DRB1*0101 allele): HA 306-318 (PKYVKQNTLKLAT); DR3 (DRB1*0301 allele):MT216 (AKTIAYDEEARRGLE); DR4 (DRB1*0401 allele): HA 306-318(PKYVKQNTLKLAT); DR7 (DRB1*0701 allele): YKL (AAYAAAKAAALAA); DRB1*1101:HA 306-318 (PKYVKQNTLKLAT); DR13 (DRB1*1301 allele): B1 21-36(TERVRLVTRHIYNREE); DR15 (DRB1*1501 allele): A3 152-166(EAEQLRRAYLDGTGVE); DRB4 (DRB4*0101 allele): E2/E7 (AGDLLAIETDKATI); andDRB5 (DRB5*0101 allele): HA 306-318 (PKYVKQNTLKLAT).

Results

Binding and non-binding peptides were first discriminated on the basisof an upper 1000 nM threshold as it is generally described in theliterature (Southwood et al (1998). J Immunol 160:3363; Geluk et al(1998) Proc Natl Acad Sci USA 95:10797), but are additionally assessedby comparison to reference peptides. The reference peptides are selectedfrom among the best binding peptides of each given HLA molecule.Relative to the reference peptides, a peptide is a weak binder for agiven HLA molecule if it has an IC50 more than 100 fold lower than thereference peptide for the given HLA molecule. A peptide is a moderatebinder is it has an IC50 more than 20 fold lower but less than a 100fold lower than the reference peptide for the given HLA molecule. Apeptide is a strong binder if it has an IC50 less than 20 fold lowerthan the reference peptide for the given HLA molecule.

Analysis of Preferred Peptide Mixtures

The nine HLA alleles used for these experiments encompass a highproportion of the Caucasian population. (Reference frequencies of HLAalleles in the population are provided in Table 3 of Example 2).Accordingly, combinations of peptides were evaluated to determine whichwould give the broadest coverage of different HLA molecules. The targetcriteria for a mixture was therefore defined as follows: For a given HLAmolecule, a mixture must comprise either 2 strong binding peptides and 1moderate binding peptide, or 1 strong binding peptide and 3 moderatebinding peptides. Preferred mixtures achieve these criteria for all ninetested HLA types. Only the peptides with sequences corresponding to SEQID NOS: 1 to 12 were considered in this analysis as peptides withsequences corresponding to SEQ ID NOS: 13 to 16 were found to be poorlysoluble. From SEQ ID NOS: 1 to 12 there are over 3000 possiblecombinations of peptides which could potentially fulfill the targetcriteria set out above.

To enable visualization of these combinations, a binary scoring systemwas applied such that for each HLA type, where a combination of peptidesachieves one of the above criteria a score of “1” was entered and wherethe criteria were not met a score of “0” is entered. The scores acrossall HLA types are then added up, such that a mixture which fulfills thecriteria for none of the HLA types will score 0, whereas a mixture whichfulfills the criteria for all nine HLA types scores 9. The scores foreach peptide combination are plotted in FIG. 2 A to Q. The highest scoreof nine was achieved by the 10 mixtures shown below:

FIG. 2 A point 16 MLA 01, 02, 03, 04, 05, 12, 14FIG. 2 B point 272 MLA 01, 03, 04, 05, 07, 12, 14FIG. 2 C point 472 MLA 02, 03, 04, 05, 06, 12, 14FIG. 2 C point 482 MLA 02, 03, 04, 05, 07, 12, 14FIG. 2 C point 488 MLA 02, 03, 04, 05, 11, 12, 14FIG. 2 C point 494 MLA 02, 03, 04, 05, 12, 14, 15FIG. 2 C point 495 MLA 02, 03, 04, 05, 12, 14, 16FIG. 2 D point 699 MLA 03, 04, 05, 07, 12, 14, 15FIG. 2 D point 700 MLA 03, 04, 05, 07, 12, 14, 16FIG. 2 G point 1271 MLA 02, 03, 04, 05, 12, 14

Thus theses mixtures are preferred combinations of peptides for use invaccination.

Example 2 Cross-Sectional Screening of Cat Allergic Subjects for T CellResponses and Basophil Histamine Release by Fel d 1-Derived, MHCCharacterised T Cell Peptide Epitopes 1. INTRODUCTION

1.1 Histamine release assay

The purpose of this assay was to identify individual peptides that arecapable of activating blood basophils (as a surrogate for tissue mastcells) resulting in histamine release that may result in allergicreactions during therapy. Peptides or combinations of peptides thatinduce histamine release frequently may be considered unsuitable forinclusion in the peptide vaccine.

Histamine release requires the crosslinking of adjacent specific IgEmolecules on the surface of the basophil. The peptides being evaluatedwere small (13 to 17 amino acids in length) and should not, therefore,possess significant tertiary structure that would enable them to retainthe conformation of an IgE-binding epitope of the whole molecule.Furthermore, peptide monomers in solution, even if they are hound byIgE, should not be able to crosslink adjacent IgE molecules. It shouldbe noted however, that some of the peptides contain cysteine residuesthat may result in disulphide bond formation between single peptides andalso between different peptides in a mixture. Thus, dimers of peptidesmay be generated that may have IgE crosslinking potential In the presentanalysis, no excipients were used in peptide formulation to prevent orreduce dimer formation through disulphide linkage.

Histamine release from fresh peripheral whole blood from cat allergicsubjects was evaluated. Peripheral blood basophils were used as asurrogate for tissue mast cells which were not practical to assay. Bloodwas incubated in vitro with 9 individual peptides from the sequence ofthe major cat allergen Fel d 1 (SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 11 and12). These peptides were selected as potential T cell epitopes followingpeptide-MHC binding assays as explained in Example 1. Additionally,responses to a preferred mixtures of a mixture of 7 peptides identifiedin Example 1 were analysed. The tested preferred mixture of 7 peptidesconsisted of the peptides of SEQ ID NOS: 1 to 7. Histamine release inresponse to whole cat dander allergen extract acted as a positivecontrol.

1.2 Proliferation Assay

The purpose of the proliferation assay was to determine the percentageof the population that responded to each individual peptide/back-uppeptide and the preferred mixture of 7 peptides.

1.3 Cytokine Assays

The purpose of the cytokine assays was two-fold; (1) to determine thepercentage of the population that responded to each individual peptideand the preferred mixture of 7 peptides, and (2) to identify individualpeptides possessing intrinsic Th2 (IL-13)-inducing characteristics whichwould be undesirable in a peptide vaccine for allergic disease, and alsoto identify individual peptides possessing intrinsic IL-10-inducingcharacteristics which may be beneficial for a peptide vaccine forallergic disease.

2. MATERIALS AND METHODS 2.1 Isolation of Peripheral Blood MononuclearCells

Peripheral blood mononuclear cells (PBMC) were isolated from theheparinised blood sample obtained from the subject. PBMC's were isolatedby Ficoll-Hypaque density gradient separation. Once isolated, the cellswere used in the cell proliferation assay, histamine release and ELISAassay and the cytokine release assay.

2.2 Histamine Release Assay and Histamine ELISA

Assays were performed on PBMC (which contain basophils). Each peptideand combinations of peptides was compared with whole allergen moleculesin a histamine release assay. Histamine concentrations were measured byELISA.

The assay required 3×10⁶ PBMC's per subject. The assay was performedusing the Immunotech Histamine Release Immunoassay kit according to themanufacturer's instructions. Following the histamine release assay,acylated samples were tested by histamine ELISA. The histamine ELISAused 50 μl of the 100 μl acylated sample generated by the histaminerelease assay. The remaining 50 μl of sample was retained, by freezingat −20° C. until the data analysis section of the ELISA has beencompleted. Once the results had been analysed and the ELISA performed ina satisfactory manner, the samples were discarded.

Peptides were assayed for their ability to induce histamine release overa 5 log₁₀ range (10 μg/ml to 1 ng/ml). The concentration range assayedwas selected based on theoretical in vivo doses of peptide that may beachieved during therapy. For example, a 10 μg dose of peptide entering ablood volume of 5 litres, would result in a blood concentration of 2ng/ml (2×10⁻⁶ mg/ml), at the lower end of the histamine release assaydose range. Whole cat dander extract (C.B.F. LETI) was used as apositive control for release over a slightly higher concentration range(100 μg to 10 ng/ml). Single measurements (i.e. not duplicate ortriplicate) were performed for each dilution. One duplicate blood samplewas assayed for spontaneous histamine release and the mean value ofthese samples was subtracted from all peptide/allergen results.

After completion of the histamine ELISA, individual histamine levelswere determined by interpolation for the standard curve generated in theELISA assay. Results from samples were adjusted to allow for anydilution of the samples. Where two or more dilutions of apeptide/allergen preparation elicited 10% or more histamine releaseabove background, or where a single value of 10% or more abovebackground was achieved at the highest concentration tested, this wasconsidered a “positive histamine release”.

2.3 Cell Proliferation Assay

The cell proliferation assay was performed on PBMC's (140×10⁶ cellsrequired for all parameters to be tested). Proliferation was measured bythe incorporation of the radiolabelled compound ³H-thymidine.

In more detail, 100 μl of the appropriate antigen or peptideconcentration was distributed into the appropriate wells of 96 wellplates. The plates were then placed into a humidified 5% CO₂ incubatorset at 37° C. for a maximum of 4 hours. PBMC's isolated as describedabove were prepared to a concentration of 2×10⁶ cells/ml in completemedium at room temperature. 100 μl of cell solution was then distributedinto each of the wells of the 96 well plates containing antigen/peptide.The plates were then incubated for 6 to 8 days. The cultures were pulsedwith tritiated thymidine solution by adding 10 μl of tritiated thymidinestock solution (1.85 MBq/ml in serum-free RPMI medium) to each well. Theplates were then returned to the incubator for between 8 and 16 hours.Cultures were then harvested using a Canberra Packard FilterMate 196cell harvester. Dried filter mats were counted using an appropriate betascintillation counter.

Counts from wells containing peptide were compared statistically towells containing media alone (12 wells per group). The non-parametricMann-Whitney test was used. The same statistical test was used for allsubjects. A statistically significant difference between media onlywells and peptide-stimulated wells was considered a positive stimulationof PBMC's by the peptide.

2.4 Cytokine Release Assay

Cytokine secretion profiles from PBMC's was analysed in response to thepeptide stimulation. Supernatants from the cytokine release assay weretested for the presence of 3 cytokines, IFN-γ, IL-10 and IL-13, usingELISA assays.

The cytokine release assay required 40×10⁶ PBMC's per subject. In moredetail, 250 μl of a 200 μg/ml solution of the appropriate antigen orpeptide concentration was distributed into the appropriate wells of 48well plates. Plates were the incubated in a humidified 5% CO₂ incubatorat 37° C. for a maximum of 4 hours. 250 μl of a 5×10⁶ cell/ml PBMCsuspension was then added to each well and the plates returned to theincubator for 5 days.

Following stimulation, samples of culture supernatant were harvestedinto 3 aliquots and frozen until the ELISA assays could be performed.One aliquot was tested for the presence of one cytokine (therefore all 3aliquots were required to test for the 3 cytokines). The cytokine levelsin the samples were determined by interpolation from standard curvesalso generated in the assay.

3. RESULTS Results Overview. 3.1 Histamine Release

TABLE 1 Histamine Release Overview Positive control Individual peptidePeptide mixture (release 10% (release 10% (release 10% above baseline)above baseline) above baseline) Highest Highest Highest 2 or more conc 2or more conc 2 or more conc Subject dilutions only dilutions onlydilutions only % age 70.4 7.4 17.3 18.5 2.5 2.5 of subjects showingrelease Total 77.8 30.9* 5 per- centage showing hista- mine release pergroup *in some subjects some peptides caused release at 2 or moreconcentrations and others at the highest dose only. Thus the two numberscannot simply be added as they are for cat dander extract and thepeptide mixture. Similarly, values for individual peptide release cannotbe added to values for the mixture of peptides since 2 of the subjectswith histamine release to the mixture also had release to individualpeptides.

Histamine release from peripheral blood basophils was observed inresponse to both positive control and peptides. Table 1 shows thepercentage of individuals in which histamine release (as defined by theacceptance criteria) occurred. Histamine release to one or moreindividual peptide occurred frequently but this rarely translated intohistamine release from the mixture of 7 preferred peptides. However, atotal of 5% of individuals displayed histamine release in response tothe peptide mixture. The details of dose and number of consecutive dosesof peptide mixture that elicit release of histamine are relevant to theinterpretation of these results and are discussed in more detail below.

TABLE 2 Individual Peptide Histamine Release Overview MLA01 (RelatedMLA03 MLA04 MLA05 MLA07 MLA12 MLA14 to SEQ (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID ID NO: 1) NO: 2) NO: 3) NO: 4) NO: 5) NO: 6) NO: 7)MLA15 MLA16 4 6 6 1 6 6 2 7 11Table 2 shows the number of individuals in whom histamine release wasdetected in response to each individual peptide. MLA15 and MLA16 mostcommonly released histamine.

3.2 Proliferation Assay Overview

FIG. 3 summarises proliferative responses to peptides and antigens. Thepercentage of individuals mounting a detectable proliferative responseis shown in the black bars. Grey (weak), white (moderate) and hashed(strong) bars provide a breakdown of the quality of these responses.Quality is arbitrarily defined by Stimulation Index (SI: ratio of countsin the presence of antigen/peptide divided by counts in medium alone).Thus for peptide 1 (MLA01), 12% of subjects made a proliferativeresponse and of these 92% were weak, none were moderate and 8% werehigh. Proliferative responses to individual peptides/antigens werevariable (black bar). 92% of subjects had positive proliferativeresponses to the positive control antigen PPD. The majority of thesewere strong responses (hashed bar). 75% of subjects responded to catdander extract, with 59% of the responses (i.e. 59% of the 75%) beingweak. The response to the mixture of 7 preferred peptides (SEQ ID NOS: 1TO 7) was almost identical to cat dander extract (CAT). Peptides MLA15and MLA16 induced more frequent responses that four of the preferredpeptides. However, MLA15 and MLA16 induced the most frequent basophilhistamine release responses (see section 3.1). Few individual peptidesinduced strong proliferative responses as expected (low precursorfrequency of peptide-specific precursor T cells).

3.3 Cytokine Assay Overview

FIG. 4 summarises the percentage of individuals who mounted a detectableresponse to each of the peptides/antigens by production of the threecytokines measured. The black bars represent production of IFN-γ, thegrey bars IL-13 and the white bars IL-10. The positive control antigenPPD elicited a cytokine production in almost all individuals (IFN-γ:91%, IL-13: 97% and IL-10: 96%). Whole cat allergen and the mixture of 7peptides elicited a cytokine response in approximately 80% or more ofsubjects. Individual peptides elicited responses of differing frequency.In general cytokine production appeared to be a more sensitive method ofdetecting responses with larger percentages of individuals givingpositive cytokine responses than proliferative responses. In most cases,IL-10 secretion was detected in the largest number of subjects and IFN-γdetected least frequently.

3.4 Tissue Typing

TABLE 3 DRB1 1 3 4 7 8 11 12 13 14 15 16 % 6.4 14.7 15.7 8.8 3.4 8.3 3.914.7 2.9 17.6 2.5 Reference 9.4 11.1 12.8 13.2 3.7 13.4 2.3 10.2 3.210.7 3.6 population %

Tissue typing was performed in order to ensure that the study population(predominantly Caucasian) was representative of the general Caucasianpopulation in which the vaccine will be used. Eleven common DRB1 allelefamilies are shown. Allele frequencies in 102 typed study subjects areshown, not the percentage of individuals expressing an allele, sinceeach individual has two DRB1 alleles and some individuals are homozygousfor particular alleles. Reference population allele frequencies are alsoshown for comparison (Data from HLA Facts Book, Parham & Barber).Reference frequencies were obtained by analysis of multiple studiesreporting frequencies and the figures shown are mean values. All of thefrequencies detected in the current analysis were within the rangesreported in the reference data. Therefore the population examined in thecurrent study is representative of a Caucasian population.

4. FIGS 4. Histamine Release Assay

TABLE 4 Individual Subject Profiles. Positive control Individual peptidePeptide mixture Spontaneous (release 10% above (release 10% above(release 10% above Release baseline) baseline) baseline) (between 2 ormore Highest 2 or more Highest 2 or more Highest Subject 10%-20%)dilutions conc only dilutions conc only dilutions conc only Comments:002 Y N N N N N No release 007 Y N Y N N N MLA16 008 Y N N N N N Norelease 009 Y N N N N N No release 010 Y N N N N N No release011_(REJECT)   30% HIGH SPONTANEOUS RELEASE 012 Y N N N N N No release013 Y N N N N N No release 014 Y N N N N N No release 015   13% N Y Y NN N MLA01, MLA04, MLA07, MLA15 016   12% Y N N N N N No release 017 Y NN N N N No release 018 N Y N N N N No release 019_(REJECT)   47% HIGHSPONTANEOUS RELEASE 020_(REJECT)   77% HIGH SPONTANEOUS RELEASE 022 Y NY N N N MLA01 023 Y N N N N N No release 024 N N N N N N No release (nopositive) 026 Y N N N N N No release 027 Y N N N N N No release 028 Y NN N N N No release 029 N N N N N N No release (no positive) 030 Y N N NN N No release 031 Y N N Y N N MLA03, MLA12 032_(REJECT)   32% HIGHSPONTANEOUS RELEASE 033 Y N N N N N No release 034 N N N N N N Norelease (no positive) 035 N Y N N N N No release 040 N Y N N N N Norelease 041 N N N N N N No release (no positive) 042 Y N N N N N Norelease 043 N N Y N N N MLA04 (no positive)

Y N N N N Y MIX 28% 046 N N Y N N N MLA16 (no positive) 047 Y N N N N NNo release 049 N N N Y N N MLA03 MLA16 (no positive) 050 Y N N N N N Norelease 051 Y N YMLA12 YMLA16 N N MLA12, MLA16 052 N Y N Y N N MLA16 053N Y N N N N 054_(REJECT)   54% UNITERPRETABLE RESULT, HIGH BACKGROUND OF40-50% RELEASE

Y N N N Y N MIX RELEASE AT 2 CONSECUTIVE MIDDLE CONCENTRATIONS

Y N Y_(MLA16) Y_(MLA03) Y N MIX RELEASE AT 4 CONSEC CONCS 057 N N Y N NN MLA15 JUST ABOVE THE 10% CUT OFF (no positive) 058 N N Y N N N MLA04(no positive) 059 Y N Y N N N MLA12 AND MLA16 060 N N N N N N NO RELEASE( NO POSITIVE) 061 Y N N N N N NO RELEASE 062 Y N Y_(MLA03)Y_(MLA01, MLA15) N N MLA03 MLA01 063 Y N N N N N NO RELEASE 064_(REJECT)  33% HIGH SPONTANEOUS RELEASE 065 Y N N N N N NO RELEASE 066 Y N N N NN NO RELEASE 067 Y N N N N N NO RELEASE 068 N N N N N N NO RELEASE (NOPOSITIVE) 069 Y N N Y N N MLA03, MLA05, MLA07 MLA12 070 Y NY_(MLA04,14,15) Y_(MLA07,12,16) N N MLA04, MLA07, MLA12, MLA14, MLA15,MLA16 071 Y N N N N N NO RELEASE 072 Y N N N N N NO RELEASE 073 Y N N YN N MLA03 076 N N N N N N NO RELEASE (NO POSITIVE) 080 Y N N N N N NORELEASE 081 N N N N N N NO RELEASE (NO POSITIVE) 082 Y N N Y_(MLA16) N NMLA16 083_(REJECT)   39% UNINTERPRETABLE ASSAY 084_(REJECT)   37%UNINTERPRETABLE ASSAY (VERY HIGH BACKGROUND) 085 Y N N N N N NO RELEASE086 N N N N N N NO RELEASE (NO POSITIVE) 087 N N N N N N NO RELEASE (NOPOSITIVE) 088 Y N N N N N NO RELEASE 089 Y N N N N N NO RELEASE 090 Y NY_(MLA03) Y_(MLA07) N N MLA03, MLA07 091 Y N N N N N NO RELEASE 09210.6% Y N N N N N NO RELEASE 093_(REJECT)   29% HIGH SPONTANEOUS RELEASE094 Y N N N N N NO RELEASE 095 Y N N N N N NO RELEASE 096 Y N N N N N NORELEASE 097_(REJECT) UNINTERPRETABLE ASSAY (APPEARS TO BE A FALSELY LOWTOTAL RELEASE COUNT) 099 Y N N N N N NO RELEASE 100 11.5% Y N Y N N NMLA15 101 N N N Y N N MLA07, MLA14, MLA15, MLA16 NO POSITIVE

Y N N Y N Y MLA01, MLA04, MLA15, MIX 104 Y N N Y N N MLA012 105_(REJECT)  28% HIGH SPONTANEOUS RELEASE 106_(REJECT)   41% HIGH SPONTANEOUSRELEASE 107_(REJECT)   35% HIGH SPONTANEOUS RELEASE 108 Y N N N N N NORELEASE 109 Y N N N N N NO RELEASE 111 Y N N Y N N MLA07 112 Y N N Y N NMLA04, MLA16 113 N N N N N N NO RELEASE (NO POSITIVE) 117 N N N N N N NORELEASE (NO POSITIVE) 118 Y N N N N N NO RELEASE % age of 70.4 7.4 17.318.5 2.5 2.5 subjects showing release MIX: mixture of 7 peptides (i.e.SEQ ID NOS: 1 to 7)

Comments: this column lists the individual peptides giving rise tohistamine release/other relevant comments for each subject.

A total of 94 histamine release assays were completed during the study.Of these 13 assays were rejected, mainly due to unacceptably high levelsof spontaneous release. Assays with spontaneous histamine release of 20%or more of the total histamine release were rejected. Those assays witha spontaneous release of between 10% and 20% are indicated in Table 4.All other assays had spontaneous release values of less than 10%.

Approximately 78% of the subjects assayed demonstrated positivehistamine release to the sensitising allergen. Existing literaturereports 10-20% of allergic individuals being resistant toallergen-induced basophil histamine release.

Histamine release was considered positive if (a) the highestconcentration of peptide alone induced release of 10% or more of thetotal release value or (b) if two consecutive values were 10% or more ofthe total release. Approximately 31% (25/81) of subjects showedhistamine release to one or more individual peptide. Of these, 6/81(7.4%) had not positive control release to whole cat allergen extract.

In two individuals the mixture of 7 peptides also induced histaminerelease in addition to certain individual peptides. In two furtherindividuals, only the mixture of 7 peptides induced release. Thus, 4/81individuals (˜5%) displayed histamine release with the mixture ofpeptides.

Subject 044 showed release (28% of total release) at the highestconcentration (10 ug/ml) of peptide only. Subject 055 showed release at0.1 ug/ml (72% of total) and 1 ug/ml (47% of total) only. Subject 056showed release at 0.01 ug/ml (11%), 0.1 ug/ml (12%), 1.0 ug/ml (17%) and10 ug/ml (10 ug/ml). Subject 103 showed histamine release (33%) at thehighest concentration (10 ug/ml) of peptide only.

4.2 Proliferation Assay

For the proliferation assay, individual proliferation data for allsubjects and all peptide concentrations was analysed. Stimulationindices to each peptide/antigen were summarised for the entirepopulation of 100 subjects.

Complex antigens such as cat dander extract and PPD induce significantproliferative responses in the population as a whole. The peptides thatinduce significant responses are those that elicit proliferativeresponses in a larger percentage of the population.

Stimulation indices of less than 1 arise when counts in wells containingpeptides are lower than those containing culture medium alone. Such aneffect may be attributable to slight changes in pH upon the addition ofpeptides which are prepared in acid solution. The absence of aproliferative response to the peptide would then result in countsslightly lower than those in the medium alone wells.

4.3 Cytokine Release Assay

FIGS. 4 to 7 show, for each peptide/antigen, the percentage ofindividuals who made a response of any detectable magnitude (i.e.production of detectable IFN-γ, IL-13 or IL-10). The strength of thoseresponses is then split into four levels of cytokine production. Forexample, 35% of the study population may have made an IFN-γ response. Ofthat 35% of individuals, half (50%) made a very weak response, 20% aweak response, 15% a moderate response and 15% a strong response (givinga total of 100% of the responders). The boundaries of each cytokinelevel were arbitrarily assigned based on the detection range of theELISA assay. The boundaries are different between IFN-γ/IL-10 and IL-13since for IFN-γ and IL-10 the detection range was approximately 1-100pg/ml whereas the range for the IL-13 assay was approximately 0.5-50pg/ml.

4.3.1 Interferon-γProduction

FIG. 5 shows the percentage of individuals producing IFN-γ and thestrength of the response following cell culture with peptide/antigen.IFN-γ responses were detected between 26-44% of subjects in response toindividual peptides. These responses were predominantly very low to lowto moderate. Complex antigens induced more frequent responses (peptidemixture 80%, cat dander 79%, PPD 91%). These responses were low tomoderate to high. PPD responses were particularly high (89 of PPDresponses were above 100 pg/ml).

4.3.2 IL-13 Production

FIG. 6 demonstrates the percentage of individuals producing IL-13 andstrength of the response following cell culture with peptide/antigen.IL-13 responses were detected in between 33-68% of subjects in responseto individual peptides. These responses were predominantly very low tolow, although a significant number of moderate responses were detected.This may reflect the Th2 nature of allergic sensitisation in thesesubjects. Complex antigens induced more frequent responses (peptidemixture 85%, cat dander 93%, PPD 97%). These responses were low tomoderate to high.

4.3.3 IL-10 Production

FIG. 7 demonstrates the percentage of individuals producing IL-10 andstrength of the response following cell culture with peptide/antigen.IL-10 responses were detected in between 46-75% of subjects in responseto individual peptides. These responses were predominantly very low tolow. Complex antigens induced more frequent responses (peptide mixture93%, cat dander 96%, PPD 96%). These responses were low to moderate.Very few “high” IL-10 responses were observed.

5. DISCUSSION 5.1 Histamine Release Assay

In interpreting the histamine release results it is important toconsider several points relating to the assay design:

-   -   1) The estimated blood dose of peptides that will be achieved        during treatment lies towards the bottom of the dose response        curve employed in the assay. For example, a 10 ug dose of        peptide entering a blood volume of 5 litres, would result in a        blood concentration of 2 ng/ml (2×10⁻⁶ mg/ml; this assumes that        no peptide is degraded which is unlikely). This concentration is        just above the lower dose limit of the assay (1 ng/ml). The 2        lowest concentrations of peptide used in the assay correspond        approximately to injected doses of 5 μg (1 ng/ml) and 50 μg (10        ng/ml). Thus, the assay is designed to detect histamine release        at or above doses of peptide used for therapy. In only 3        instances was histamine release associated with the lowest two        (consecutive values above 10%) concentrations of peptide. In two        of these cases values were less than 11%. The 7 peptide mixture        did not show any release at the lowest 2 concentrations of        peptide. Thus, although histamine release in response to        individual peptides or the mixture was relatively common, it was        generally not seen at the concentrations of peptide that will be        achieved during therapy.    -   2) For reasons of cost and complexity, only single wells were        assayed for each concentration of peptide. This increases the        risk that any one value may be spurious. This is particularly        relevant to the second condition defined for a positive result;        that the highest concentration alone of peptide/antigen shows        release of 10% or more of the total release. Several cases of        histamine release to individual peptides were only associated        with the single highest concentration of peptide and this was        also true for 2/4 individuals with histamine release triggered        by the mixture of 7 peptides.    -   3) In some cases, histamine release from peptides was not        associated with histamine release from cat dander extract        (absence of positive control).    -   4) Peptides with cysteine residues (MLA01, MLA04, MLA05, MLA12        and MLA15) were previously shown to be capable of varying        degrees of homo-dimerisation. Although not formally quantified,        these peptides when mixed are likely to also form hetero-dimers        (i.e. within the SEQ ID NOS: 1 TO 7 mixture). Dimers may be        sufficient to crosslink IgE molecules on the surface of mast        cells and basophils giving rise to histamine release. No        excipients to reduce disulphide bond formation between        homologous of heterologous peptides were used in this study.        Clinical preparations of the vaccine will contain thioglycerol        to block disulphide bond formation.

Approximately 78% of the subjects assayed demonstrated positivehistamine release to the sensitising allergen. This is slightly lowerthan reports in the literature which suggest that 10-20% of allergicindividuals are resistant to allergen-induced basophil histaminerelease.

30.9% of subjects showed histamine release to one or more individualpeptide. Histamine release was also detected in 5% of subjects (4/81) tothe mixture of 7 peptides (SEQ ID NOS: 1 TO 7; likely vaccinecandidates). Two of these 4 individuals displayed release to individualpeptides and 2 did not. In several subjects showing histamine release toindividual peptides (6/81; 7.4%), release only occurred with peptideMLA15 or MLA16, which are not included in the SEQ ID NOS: 1 TO 7mixture. MLA16 was the peptide most frequently associated with histaminerelease. Adjusting values for individual peptide release to include onlythose peptides in the preferred 7 vaccine candidates, 23.5% of subjectsdisplayed histamine release to individual components of the vaccine.

5.2 Proliferation Assay

Proliferation of PBMC was assayed in response to culture with 3concentrations of individual peptides, a mixture of 7 peptides (selectedby MHC binding assays) and whole cat dander allergen extract. Responsesto PPD at a single concentration were also measured as a marker of apositive recall response.

PPD responses: 92% of subjects mounted a detectable proliferativeresponse to PPD. The response is largely dependent upon priorvaccination with BCG. Non-responders may have originated from countriesin which BCG is not mandatory (e.g. USA), or may not have received theimmunisation for other reasons. The majority of responses (92%) resultedin an SI of greater than 10. These were arbitrarily assigned as “strong”responses.

Cat dander allergen extract responses: 75% of subjects mounted adetectable proliferative response to cat dander allergen extract. Morefrequent responses were detected through measurement of cytokineshighlighting the importance of assaying multiple parameters ofactivation to determine reactivity. The majority of responses were weak(SI 2-5; 59%) although significant numbers of moderate (SI 5-10; 24%)and strong (SI 10+; 17%) were observed.

Peptide mixture (P1-7): 71% of subjects mounted a response to thepeptide mixture, similar to cat dander allergen extract. A similarpercentage of weak (52%), moderate (34%) and strong (14%) responses wereobserved. Proliferative responses to cat dander allergen extract andpeptide mixture correlated closely indicating that the majority of Tcell reactivity to cat dander can be accounted for by the epitopescontained within the peptide mixture.

Individual peptide responses: Proliferative responses to individualpeptides were generally weak to moderate. Most peptides generated 70-80%of their responses in the weak category with 20-30% in the moderatecategory. Few peptide elicited strong responses. Weaker responses toindividual peptides than to complex antigens or mixtures of peptides isan expected finding resulting from lower precursor frequencies of Tcells specific for individual epitopes.

The strongest proliferative responses to an individual peptide were toP12 from Fel d 1 chain 2 (43%) and the weakest to P4 from chain 1 (6%).However, cytokine responses to all peptides were detected morefrequently than proliferative responses.

5.3 Cytokine Assays

Cytokine measurement proved to be the most sensitive method of measuringresponses to the peptides. Generally a higher percentage of subjectsdisplayed measurable cytokine responses compared to measurableproliferative responses. Production of each of the three cytokinesvaried with IL-10 generally being produced by a greater proportion ofsubjects than IL-13 and IFN-γ. The lowest frequency of response wasdetected with IFN-γ. The atopic allergic status of these subjects islikely to mean that the memory T cell response to Fel d 1 and itsepitopes will be dominated by Th2 responses which may account for theless frequent Th1 (IFN-γ) response. The high frequency of IL-10responses was a surprise. IL-10 is considered to be a Th2 cytokine inthe murine system but this is not well established in the human system.IL-10 is generally regarded as a regulatory/immunosuppressive cytokine.Previous reports have suggested that some peptide sequences may haveintrinsic IL-10 inducing properties. Such peptides were not observed inthis study. The detection of such responses in other systems may simplyreflect the nature of T cell priming to whole allergen which is recalledby culture of memory T cells with peptide. Thus, production of IL-10 maybe a recall response rather than the result of intrinsic IL-10-inducingcharacteristics of the peptide.

No single peptide induced the preferential production of a particularcytokine. Thus, none of the peptides screened induced a particularlyunfavourable Th2 (IL-13) response which would have been consideredundesirable for inclusion in the peptide vaccine.

5.4 Tissue Typing

Tissue typing results show that a representative population was assayedin this study.

6. CONCLUSION 6.1 Histamine Release Assay

Individual peptides induced histamine release in some individuals. Themixture of preferred peptides SEQ ID NOS: 1 TO 7 induced histaminerelease in 4 individuals although in 2 of these the release was detectedat a single point (highest concentration). MLA16 caused most frequentrelease but is absent from SEQ ID NOS: 1 TO 7. Some positive release wasobserved with peptides in the absence of “positive control release” fromwhole cat dander. The assay was designed to detect histamine release atconcentrations of peptide approximating to treatment doses and above.Histamine release at concentrations of peptide corresponding totreatment doses was extremely rare (only one clear example) and onlyoccurred with individual peptides, not with SEQ ID NOS: 1 TO 7.

The results of the in vitro histamine release assay are likely toover-represent the histamine releasing potential of the vaccine since nosteps were taken to minimise disulphide bond formation between peptides.

Histamine was released by basophils from the majority of individuals inthe presence of whole cat dander extract. Histamine release occurred ina dose-dependent fashion in many subjects in contrast to release withpeptides which frequently occurred at concentrations in the middle ofthe dose range. In individuals where histamine was released by peptides,sensitivity to cat dander extract was usually apparent at lower doses ofextract.

6.2 Proliferation Assay

Proliferative responses to peptides were weaker than to peptide mixturesor complex protein antigens as expected. Most individual peptideelicited proliferative responses in less than 20% of individuals.Considerable variation was seen between peptides but no single peptidefailed to elicit proliferative responses in at least some subjects,although one of the preferred 7 peptides MLA04 was poor at inducingproliferation. Peptides MLA15 and MLA16 were more potent in induction ofproliferation than several of the preferred 7 peptides but gave thehighest histamine release.

6.3 Cytokine Assays

Cytokine production was a more sensitive method than proliferation fordetecting responses to peptides in this study. No evidence was obtainedto support the idea that certain peptides may have an intrinsic abilityto induce a particular pattern of cytokine production. No single peptidepreferentially elicited a Th1, Th2 or Treg (IL-10) response. IFN-γresponses tended to be less common than IL-13 and IL-10. The cytokineassay data does not indicate that any of the preferred peptide mixturebe substituted nor that any single peptide or the mixture willpreferentially induce a Th2 response in vivo.

Example 3 Clinical Trial of Preferred Combination

A preferred mixture of 7 peptides consisting of the peptides of SEQ IDNOS: 1 to 7 has been tested in a randomised, placebo-controlled, blindclinical trial. The efficacy of this mixture in reducing allergicsymptoms was evaluated. The study design of the clinical trial was inaccordance with good clinical practice guidelines.

Baseline skin responses to cat allergen for all subjects wereestablished using a Baseline Challenge which took place between 6 and 8days prior to study medication administration. Two intradermalinjections of 0.010 HEP (histamine equivalent prick) units ofcommercially available standard cat allergen (supplied by LaboratoriosLeti, Spain) were administered, separated by a 30 minute time interval,into the volar surface of the left and right forearms respectively.Subjects were assessed to ensure that they experience a Late-Phase SkinResponse (LPSR) to whole cat allergen, and the magnitude of the baselinereaction was recorded as follows:

Eight hours after each injection the outline of any late-phase responsewas drawn onto the skin with a ballpoint pen. The longest and orthogonaldiameters were measured and recorded for each response, and the area ofthe response in each arm was calculated. The average area of response inboth arms of each subject was then calculated to provide the baselinereaction. Subjects who produced a suitable baseline reaction wereassigned to dosing groups, randomised and entered into the TreatmentPhase.

The Treatment Phase consisted of a period of 21 days for each subject.During this period one group of subjects received a single intradermalinjection of either the preferred mixture (0.03, 0.3, 3, 12 nmol of eachpeptide per dose) or diluent placebo at Treatment Phase Visit 1 on dayone. A cohort of 8 subjects received treatment at each dose level (6received the preferred mixture and 2 placebo). The first cohort of theintradermal group received 0.03 nmol of each peptide in the mixture andeach subsequent cohort in the group received the next higher dose level.

Intradermal injections were made into the flexor surface of the leftforearm. The total volume of the injection was 60 μL for all injections.After treatment, subjects had their skin response to whole allergenretested at Treatment Phase Visit 2 on day 21 (±3 days). Skin responsesto cat allergen were assessed by measurement of the late-phase responses8 hours following intradermal administration of 0.010 HEP (histamineequivalent prick) units of commercially available standard cat allergen(supplied by Laboratorios Leti, Spain) as described above. The averagearea of response for both arms of each subject was then calculated asdescribed above.

This average LPSR area after treatment was then compared to the baselineLPSR area for each subject. The overall change in LPSR area for alleight patients in each cohort was then evaluated. The results of thisanalysis are shown in the table below. This analysis was performedwithout unblinding the data.

REDUCTION IN LPSR AREA DOSE (nmol) FOLLOWING TREATMENT 0.03 + 0.3 ++ 3.0++ 12.0 ++FIG. 8 is a representative plot showing the average LPSR area before andafter treatment for all eight patients in the 12.0 nmol cohort. Takentogether, these data indicate that the preferred mixture of peptides iseffective at reducing the LPSR to whole allergen in cat allergicindividuals.

1-23. (canceled)
 24. A pharmaceutical formulation comprising apharmaceutically acceptable carrier or diluent, a preservative, and, asthe only therapeutic ingredients: (I) a polypeptide which consists ofthe sequence of SEQ ID NO: 1, a polypeptide which consists of thesequence of SEQ ID NO: 2, a polypeptide which consists of the sequenceof SEQ ID NO: 3, a polypeptide which consists of the sequence of SEQ IDNO: 4, a polypeptide which consists of the sequence of SEQ ID NO: 5, apolypeptide which consists of the sequence of SEQ ID NO: 6 and apolypeptide which consists of the sequence of SEQ ID NO: 7 or (II) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 10, apolypeptide which consists of the sequence of SEQ ID NO: 6 and apolypeptide which consists of the sequence of SEQ ID NO: 7; or (III) apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 5, apolypeptide which consists of the sequence of SEQ ID NO: 6, apolypeptide which consists of the sequence of SEQ ID NO: 7 and apolypeptide which consists of the sequence of SEQ ID NO: 11; or (IV) apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 5, apolypeptide which consists of the sequence of SEQ ID NO: 6, apolypeptide which consists of the sequence of SEQ ID NO: 7 and apolypeptide which consists of the sequence of SEQ ID NO: 12; or (V) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 6 and apolypeptide which consists of the sequence of SEQ ID NO: 7; or (VI) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 6, apolypeptide which consists of the sequence of SEQ ID NO: 7 and apolypeptide which consists of the sequence of SEQ ID NO: 1; (VII) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 6, polypeptidewhich consists of the sequence of SEQ ID NO: 7 and a polypeptide whichconsists of the sequence of SEQ ID NO: 5; or (VIII) a polypeptide whichconsists of the sequence of SEQ ID NO: 8, a polypeptide which consistsof the sequence of SEQ ID NO: 2, a polypeptide which consists of thesequence of SEQ ID NO: 3, a polypeptide which consists of the sequenceof SEQ ID NO: 4, a polypeptide which consists of the sequence of SEQ IDNO: 6, a polypeptide which consists of the sequence of SEQ ID NO: 7 anda polypeptide which consists of the sequence of SEQ ID NO: 9; (IX) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 6, apolypeptide which consists of the sequence of SEQ ID NO: 7 and apolypeptide which consists of the sequence of SEQ ID NO: 11; or (X) apolypeptide which consists of the sequence of SEQ ID NO: 8, apolypeptide which consists of the sequence of SEQ ID NO: 2, apolypeptide which consists of the sequence of SEQ ID NO: 3, apolypeptide which consists of the sequence of SEQ ID NO: 4, apolypeptide which consists of the sequence of SEQ ID NO: 6, apolypeptide which consists of the sequence of SEQ ID NO: 7 and apolypeptide which consists of the sequence of SEQ ID NO: 12; wherein:any polypeptide in each of (I) to (X) may be replaced by a variant orfragment of the replaced polypeptide, provided that at least four saidpolypeptides are not so replaced, wherein (i) a variant of a saidpolypeptide is up to 30 amino acids in length and comprises the sequenceof the said polypeptide; or (ii) a variant of a said polypeptide is upto 30 amino acids in length and comprises or consists of the sequence ofsaid polypeptide in which one, two, three or four amino acidsubstitutions have been made; and (iii) a fragment of a said polypeptideis at least 9 amino acids in length and comprises the sequence of thesaid polypeptide in which 1, 2 or 3 amino acids are deleted from the Nand/or C terminal ends of the said sequence.
 25. The formulationaccording to claim 24 in which any polypeptide in each of (I) to (X) maybe replaced by a variant or fragment of the replaced polypeptide,provided that at least five or six said polypeptides are not soreplaced.
 26. The formulation according to claim 24 in which anypolypeptide in each of (I) to (X) may be replaced by a variant orfragment of the replaced polypeptide, provided that the polypeptides ofSEQ ID NOS: 2, 3, 4, 6 and 7 are not so replaced.
 27. The formulationaccording to claim 24 in which no polypeptide is replaced by a variantor fragment.
 28. The formulation according to claim 24 which comprisesno further polypeptides.
 29. The formulation according to claim 24 whichis provided as an injectable solution, suspension or emulsion.
 30. Theformulation according to claim 24, wherein each polypeptide has aconcentration in the range of 0.03 to 200 nmol/ml, 0.3 to 200 nmol/ml or10 to 50 nmol/ml.
 31. The formulation according to claim 24 which isprovided in dry form for reconstitution.
 32. A method of preparing apharmaceutical formulation as defined in claim 24, comprising combiningsaid polypeptides, variants or fragments with a pharmaceuticallyacceptable carrier or diluent.
 33. A method according to claim 32 forpreparing a composition for parenteral administration comprisingproviding said polypeptides, variants or fragments in dry form andreconstituting said polypeptides, variants or fragments with a saidpharmaceutically acceptable carrier or diluent.